Luciferase-based thermal shift assays

ABSTRACT

Provided herein are systems and methods for characterizing target/ligand engagement. In particular, luciferase-labeled polypeptide targets are used to detect or quantify target/ligand engagement (e.g., within a cell or cell lysate).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/787,950, filed Feb. 11, 2020, now allowed, which is adivisional of U.S. patent application Ser. No. 15/017,271, filed Feb. 5,2016, now U.S. Pat. No. 10,571,741, which claims the priority benefit ofU.S. Provisional Patent Application 62/112,518, filed Feb. 5, 2015, eachof which is incorporated by reference in its entirety.

FIELD

Provided herein are systems and methods for characterizing target/ligandengagement. In particular, luciferase-labeled polypeptide targets areused to detect or quantify target/ligand engagement (e.g., within a cellor cell lysate).

BACKGROUND

Multiple challenges face drug development today including high costs andlong development cycles for new therapeutics. Methods that promoteaccelerated drug development are urgently needed. The efficacy oftherapeutics is dependent on a drug binding to its target. Due to itssimplicity, a protein thermal shift assay (TSA) is a commonly usedmethod for screening libraries and validating hits in drug discoveryprograms. The most common method of TSA in use today can only beperformed using purified protein, which has several disadvantages.Recently, the cellular thermal shift assay (CETSA) was developed todetect endogenous target protein within cells or cell lysates, therebyalleviating the need to create purified proteins and allowing for targetengagement analysis in complex environments that are more biologicallyrelevant. However, the CETSA is a multi-step assay in which the analysisof target engagement relies upon western blot or AlphaScreen (PerkinElmer) technologies, both of which have several disadvantages, e.g.,cost, insensitive, multi-stepped protocols, requires cell lysis and spinsteps, low throughput (e.g., Western blot), dependent upon antibodyrecognition of native protein state, cell line optimization, requires alarge amount of cells, etc. What is needed is a simple, homogeneous,rapid, and inexpensive TSA, whereby target engagement can becharacterized in complex environments, such as cells and cell lysate.

SUMMARY

Provided herein are systems and methods for characterizing target/ligandengagement. In particular, luciferase-labeled polypeptide targets areused to detect or quantify target/ligand engagement (e.g., within acell, e.g., in an intact live cell, or cell lysate).

In some embodiments, provided herein are systems comprising: (a) afusion of a target protein and a bioluminescent reporter; and (b) afluorescent dye; wherein the bioluminescent reporter and the fluorescentdye comprise a bioluminescence resonance energy transfer BRET pair(e.g., the bioluminescent reporter is the BRET donor and the fluorescentdye is the BRET acceptor); and wherein the fluorescent dye interactswith exposed hydrophobic regions of the target protein upon unfolding,denaturation, and/or aggregation of the target protein. In someembodiments, the fluorescent dye does not interact with the nativelyfolded (or otherwise stably folded) target protein. In some embodiments,the fluorescent dye binds to the unfolded or partially unfolded targetprotein significantly more than to the folded target protein (e.g.,2-fold increase, 3-fold increase, 4-fold increase, 5-fold increase,6-fold increase, 7-fold increase, 8-fold increase, 9-fold increase,10-fold increase, 20-fold increase, 50-fold increase, 100-fold increase,1000-fold increase, or more, or ranges therein). In some embodiments,systems further comprise a ligand or test ligand for the target protein.In some embodiments, systems further comprise a substrate for thebioluminescent reporter.

In some embodiments, provided herein are systems comprising: (a) afusion of a target protein and a bioluminescent reporter, wherein thebioluminescent reporter protein has an emission spectra that encompassesa wavelength X; and (b) a fluorescent dye that: (1) bindsnon-specifically to aggregated proteins and/or hydrophobic peptidesegments, and (2) has an excitation spectra that encompasses thewavelength X. In some embodiments, the emission spectra of thebioluminescent reporter protein and the excitation spectra of thefluorescent dye overlap such that the bioluminescent reporter excitesthe fluorescent dye by BRET. In some embodiments, the bioluminescentreporter and the fluorescent dye comprise a BRET pair. In someembodiments, the bioluminescent reporter is a BRET donor, and thefluorescent dye is a BRET acceptor. In some embodiments, the system is acell, e.g., a live, intact cell, cell lysate (e.g., cells lysed bychemical methods (e.g., lytic NANOGLO reagent, cells lysed bysonication, etc.), or reaction mixture. In some embodiments, thebioluminescent reporter is a luciferase. In some embodiments, theluciferase is a variant Oplophorus gracilirostris luciferase (OgLuc)(e.g., >60%, 70%, 80%, 90%, or 95% identity with SEQ ID NO: 1, SEQ IDNO: 2, etc.). In some embodiments, the bioluminescent reporter is apeptide or polypeptide tag that forms a bioluminescent complex uponinteraction with a complement polypeptide or peptide (See, e.g., U.S.Pub. No. 2014/0363375; herein incorporated by reference in itsentirety). In some embodiments, the system further comprises complementpolypeptide or peptide that forms a bioluminescent reporter with thetarget portion of the fusion. In some embodiments, provided herein arecompositions and methods for the assembly of a bioluminescent complexfrom two or more non-luminescent (e.g., substantially non-luminescent)peptide and/or polypeptide units. In particular, bioluminescent activityis conferred upon a non-luminescent polypeptide via structuralcomplementation with another, complementary non-luminescent peptide. Asused herein the term “complementary” refers to the characteristic of twoor more structural elements (e.g., peptide, polypeptide, nucleic acid,small molecule, etc.) of being able to hybridize, dimerize, or otherwiseform a complex with each other. For example, a “complementary peptideand polypeptide” are capable of coming together to form a complex. Insome embodiments, the fluorescent dye is an environmentally-sensitivedye. In some embodiments, the fluorescent dye is hydrophobic. In someembodiments, the fluorescence of the fluorescent dye is quenched bywater (e.g., fluorescence is suppressed when the dye is free in aqueoussolution and enhanced when the dye is in a hydrophobic environment). Insome embodiments, the fluorescent dye is fluorogenic. In someembodiments, binding between the fluorescent dye and the reporterprotein is altered by changes in target protein structure (e.g.,unfolding). In some embodiments, the fluorescence of the fluorescent dyeis quenched or suppressed when free in aqueous solution and enhancedwhen the dye bound to a hydrophobic composition. In some embodiments,the fluorescent dye is SYPRO Orange, SYPRO Red, Nile Red, ANS, Bis-ANS,SYPRO Ruby, SYPRO Tangerine, and/or Dapoxyl Sulfonic Acid Sodium Salt,PROTEIN THERMAL SHIFT Dye (Life Technologies), PROTEOSTAT Dye (Enzo), orcombinations thereof. In some embodiments, the system further comprisesa ligand of the target or a test ligand.

In some embodiments, methods are provided for detecting the interactionof a ligand and target protein based upon the stabilization of thetarget protein upon formation of a target/ligand complex comprising: (a)incubating a fusion of the target protein and a bioluminescent reporterwith a substrate for the bioluminescent reporter and a fluorescent dyethat binds to hydrophobic peptide sequences when they are exposed byprotein unfolding; and (b) detecting BRET from the bioluminescentreporter to the fluorescent dye in the presence and absence of theligand or test ligand. In some embodiments, signal is detected over arange of denaturing conditions (e.g., two or more conditions (e.g.,ranging from non-denaturing to highly-denaturing)). In some embodiments,due to the proximity limitation of BRET (e.g., donor to acceptordistance of about 1-10 nm), emission from the fluorescent dye as aconsequence of BRET from the bioluminescent reporter is only detectedwhen the fluorescent dye is bound to the target protein. In someembodiments, binding of the ligand or test ligand to the target proteinstabilizes the protein and increases the degree of the denaturingconditions (e.g., temperature, concentration of denaturant, increasedpressure, etc.) required to unfold the target protein, allow binding ofthe fluorescent or fluorogenic dye to the exposed hydrophobic portionsof the target protein, producing a detectable signal resulting from BRETfrom the bioluminescent reporter to the target-bound fluorescent dye. Insome embodiments, the fusion, fluorescent dye, substrate, and ligand(when present) are combined in any suitable order of addition.

In some embodiments, provided herein are methods to detect atarget/ligand interaction, comprising the steps of: (a) incubating afusion of a target protein and a reporter polypeptide: (i) in thepresence of a ligand to produce a test sample and (ii) in the absence ofa ligand to produce a control sample; (b) treating said test and controlsamples under conditions that cause the target protein to unfold (e.g.,to an appropriate extent); (c) measuring signal from the reporterpolypeptide in said test and control samples; and (d) comparing themeasurement made in step (c) between the test and control samples,wherein alteration of the signal from said reporter polypeptide in thetest sample compared to the control sample indicates the presence of atarget/ligand interaction. In some embodiments, the fusion is within acell, e.g., a live, intact cell, cell lysate, or reaction mixture. Insome embodiments, the fusion is expressed within the cell, e.g., a liveintact cell, cell lysate, or reaction mixture. In some embodiments, theligand is added exogenously to the cell, e.g., a live intact cell, celllysate, or reaction mixture. In some embodiments, the reporterpolypeptide is a luciferase. In some embodiments, the luciferase is avariant Oplophorus gracilirostris luciferase (OgLuc) (e.g., >60%, 70%,80%, 90%, or 95% identity with SEQ ID NO: 1, SEQ ID NO: 2, etc.). Insome embodiments, the bioluminescent reporter is a peptide orpolypeptide tag that forms a bioluminescent complex upon interactionwith a complement polypeptide or peptide (See, e.g., U.S. Pub. No.2014/0348747; herein incorporated by reference in its entirety). In someembodiments, the complement polypeptide or peptide is added exogenouslyto the test and control samples prior to step (c). In some embodiments,the complement polypeptide or peptide is expressed within the cell, celllysate, or reaction mixture. In some embodiments, the conditions thatcause the target protein to unfold to an appropriate extent compriseelevated temperature, increased pressure, and/or a denaturant. In someembodiments, elevated temperature comprises one or more temperaturesabove physiologic temperature for the protein in question. In someembodiments, elevated temperature comprises one or more temperaturesnear (e.g., +/−1, 2, 3, 4, 5, 10, 15, 20° C.) the approximate meltingtemperature of the target protein. In some embodiments, a plurality oftest samples is produced using a plurality of test ligands.

In some embodiments, provided herein are methods to detect atarget/ligand interaction, comprising the steps of: (a) incubating afusion of a target protein and a bioluminescent reporter: (i) in thepresence of a ligand to produce a test sample, and (ii) in the absenceof a ligand, to produce a control sample; (b) contacting the test andcontrol samples with an environmentally-sensitive hydrophobic dye,wherein the emission spectra of the bioluminescent reporter and theexcitation spectra of the environmentally-sensitive hydrophobic dyeoverlap; (c) treating said test and control samples under conditionsthat cause the target protein to unfold to an appropriate extent; (d)measuring signal from the environmentally-sensitive hydrophobic dye insaid test and control samples (e.g., emission resulting from energytransfer from the active bioluminescent reporter after addition ofsubstrate); and (e) comparing the measurement made in step (d) betweenthe test and control samples, wherein alteration of the signal from saidenvironmentally-sensitive hydrophobic dye in the test sample compared tothe control sample indicates the presence of a target/ligandinteraction. In some embodiments, steps (a), (b), and (c) are performedin any suitable order (e.g., a-b-c, a-c-b, b-a-c, b-c-a, c-a-b, orc-b-a). In some embodiments, the fusion is within a cell, e.g., a liveintact cell, cell lysate, or reaction mixture. In some embodiments, thefusion is expressed within the cell, e.g., a live intact cell, celllysate, or reaction mixture. In some embodiments, the ligand is addedexogenously to the cell, e.g., a live intact cell, cell lysate, orreaction mixture. In some embodiments, the environmentally-sensitivehydrophobic dye is added exogenously to the cell, e.g., a live intactcell, cell lysate, or reaction mixture. In some embodiments, thebioluminescent reporter is a luciferase. In some embodiments, theluciferase is a variant Oplophorus gracilirostris luciferase (OgLuc)(e.g., >60%, 70%, 80%, 90%, or 95% identity with SEQ ID NO: 1, SEQ IDNO: 2, etc.). In some embodiments, the bioluminescent reporter is apeptide or polypeptide tag that forms a bioluminescent complex uponinteraction with a complement polypeptide or peptide. In someembodiments, the complement polypeptide or peptide is added exogenouslyto the test and control samples prior to step (d). In some embodiments,the complement polypeptide or peptide is expressed within the cell,e.g., a live intact cell, cell lysate, or reaction mixture. In someembodiments, the conditions that cause the target protein to unfold toan appropriate extent comprise elevated temperature, increased pressure,and/or a denaturant. In some embodiments, elevated temperature comprisesone or more temperatures above physiologic temperature. In someembodiments, elevated temperature comprises one or more temperaturesnear the approximate melting temperature of the target protein. In someembodiments, the environmentally-sensitive hydrophobic dye bindsnonspecifically to hydrophobic surfaces. In some embodiments, theenvironmentally-sensitive hydrophobic dye binds preferentially to thefolded, unfolded, or molten globule states of the protein. In someembodiments, the fluorescence of the environmentally-sensitivehydrophobic dye is quenched by water. In some embodiments, theenvironmentally-sensitive hydrophobic dye is Sypro Orange, SYPRO Red,Nile Red, ANS, Bis-ANS, SYPRO Ruby, SYPRO Tangerine, and/or DapoxylSulfonic Acid Sodium Salt, Protein Thermal Shift™ Dye (LifeTechnologies), PROTEOSTAT Dye (Enzo), or combinations thereof. Othersuitable dyes for use in embodiments described herein include, but arenot limited to: styryl dyes, asymmetric cyanines, oxazole dyes(Dapoxyls), azo dyes, and other dyes such as Thioflavin T and(Dicyanovinyl)julolidine (DCVJ). Suitable dyes for use in embodimentsdescribed herein are also described in, for example: Kovalska et al.Dyes and Pigments 67 (2005) 47-54.; Volkova et al. Bioorganic &Medicinal Chemistry 16 (2008) 1452-1459.; Volkova et al. J. Biochem.Biophys. Methods 70 (2007) 727-733.; Hawe et al. PharmaceuticalResearch, Vol. 25, No. 7, July 2008.; U.S. Pub. No. 2011/0130305; PCTPub. WO 2006/079334; Diwu et al. Photochemistry and Photobiology, 1997,66(4): 424-431.; herein incorporated by reference in their entireties.

In some embodiments, methods of screening a group of test ligands forinteraction with a target protein are provided. In some embodiments, afusion of the target protein and a bioluminescent reporter is combinedwith a fluorescent dye that binds to exposed hydrophobic portions ofproteins (e.g., upon exposure of the hydrophobic portions due to proteindenaturation) and a substrate for the bioluminescent reporter, in thepresence and absence of one or more of the test ligands. In someembodiments, multiple assays, each comprising a different test ligand ofset of test ligands are performed in parallel (e.g., in a highthroughput method). Fluorescence emission from the fluorescent dye as aresult of BRET from the bioluminescent reporter is detected. Decrease ofBRET-induced fluorescence in the presence of the one or more testligands, and/or an increase in the temperature or amount of denaturantrequired to generate a BRET-induced fluorescent signal, indicatesinteraction of one or more of the test ligands with the target protein.

In some embodiments, provided herein are methods of screening a group oftest ligands for binding to a target protein comprising: (a) creating aplurality of test samples each comprising at least one test ligand and afusion of a target protein and a bioluminescent reporter; (b) creatingat least one control sample comprising a fusion of a target protein anda bioluminescent reporter in the absence of a test ligand; (c)contacting the test and control samples with anenvironmentally-sensitive hydrophobic dye, wherein the emission spectraof the bioluminescent reporter and the excitation spectra of theenvironmentally-sensitive hydrophobic dye overlap; (d) treating the testand control samples under conditions that cause the target protein tounfold to an appropriate extent; (e) exposing the test and controlsamples to the substrate of the bioluminescent reporter; (f) measuringsignal from the environmentally-sensitive hydrophobic dye in said testand control samples; and (g) comparing the measurement made in step (f)between the test and control samples, wherein alteration of the signalfrom said environmentally-sensitive hydrophobic dye in one or more testsamples compared to the control sample indicates the presence of atarget/ligand interaction.

In some embodiments, provided herein are methods to detect atarget/ligand interaction, comprising the steps of: (a) incubating afusion of a target protein and a reporter polypeptide in the presence ofa ligand to produce a test sample; (b) treating said test sample underconditions that cause the target protein to unfold to an appropriateextent; (c) measuring signal from the reporter polypeptide in said testsample; and (d) detecting target/ligand interaction based on the signalfrom said reporter polypeptide in the test sample.

In some embodiments, provided herein are methods to detect atarget/ligand interaction, comprising the steps of: (a) incubating afusion of a target protein and a bioluminescent reporter in the presenceof a ligand to produce a test sample; (b) contacting the test samplewith an environmentally-sensitive hydrophobic dye, wherein the emissionspectra of the bioluminescent reporter and the excitation spectra of theenvironmentally-sensitive hydrophobic dye overlap; (c) treating the testsample under conditions that cause the target protein to unfold to anappropriate extent; (d) exposing the test sample to the substrate of thebioluminescent reporter, (e) measuring signal from theenvironmentally-sensitive hydrophobic dye in said test sample; and (f)detecting target/ligand interaction based on the signal from saidenvironmentally-sensitive hydrophobic dye in the test sample.

Based on the successful use of a direct luciferase fusion or BRET todetect ligand-mediated thermal stabilization, other proximity-basedreporter chemistries are understood to be useful. For example, incertain embodiments, an epitope tag is attached to the protein ofinterest. Following a thermal denaturation step, addition of a detectionantibody labeled with a donor fluorophore (e.g., terbium, europium,etc.) is used in a FRET assay with a denaturation/aggregation-sensitivedye as a FRET acceptor. Ligand-mediated thermal stabilization results ina loss of FRET/TR-FRET signal. In other embodiments, a combination oflabeled antibodies (e.g. donor-labeled anti-FLAG and acceptor labeledanti-V5) and a tandem epitope (e.g. FLAG-V5) tethered to the targetprotein are used as a detection system. When stabilized, the target isstabilized by binding of a ligand, the epitope is presented and bothantibodies bind, generating a proximity-based signal (e.g. FRET,TR-FRET). Upon thermal denaturation, the epitopes are unavailable to theantibody pair, resulting in a loss of proximity-based signal. In someembodiments, various detection chemistries are applied (e.g. TR-FRET,proximity ligation, singlet oxygen transfer/alphascreen, etc.).

In some embodiments, methods are provided for the detection oftarget/ligand interactions within a cell. In some embodiments, methodsallow detection of target/ligand interactions within live, intact cells.In some embodiments, all steps (e.g., target/reporter expression,reagent (e.g., ligand, substrate, dye, etc.) addition, targetdenaturation, signal detection, etc.) are performed to or within thelive, intact cells. In some embodiments, the target/ligand interactionis initiated within the live, intact cells, but one or more steps of theassay (e.g., substrate addition, target denaturation, signal detection,etc.) are performed in a lysate of the cells (e.g., following lysis ofthe live, intact cells to produce a cell lysate). In some embodiments,all steps (e.g., target/reporter expression, reagent (e.g., ligand,substrate, dye, etc.) addition, target denaturation, signal detection,etc.) are performed to or within the cell lysate. The followingparagraphs provide exemplary methods for performing such assays. Thefollowing embodiments are not limiting, and may be combined and/ormodified with other embodiments described herein.

In some embodiments, provided herein are methods to detect atarget/ligand interaction within a live, intact cell, comprising thesteps of: (a) providing a live, intact cell expressing a fusion of atarget protein and a bioluminescent reporter, (b) allowing time for thetarget protein to interact with the ligand within the live, intact cell;(c) contacting the live, intact cell with an environmentally-sensitivehydrophobic dye, wherein the emission spectra of the bioluminescentreporter and the excitation spectra of the environmentally-sensitivehydrophobic dye overlap; (d) treating the live, intact cell underconditions that cause the target protein to unfold to an appropriateextent; (e) exposing the live, intact cell to the substrate of thebioluminescent reporter, (f) measuring signal from theenvironmentally-sensitive hydrophobic dye and bioluminescent reporter inthe live, intact cell; and (g) detecting target/ligand interaction basedon the ratio from the signals from said environmentally-sensitivehydrophobic dye and bioluminescent reporter in the live, intact cell. Insome embodiments, methods further comprise a step of adding the ligandto the live, intact cell. In some embodiments, the ligand is endogenousto the live, intact cell. In some embodiments, the signal from saidenvironmentally-sensitive hydrophobic dye in the live, intact cell iscompared to a control sample without the ligand.

In some embodiments, provided herein are methods to detect atarget/ligand interaction within a cell, comprising the steps of: (a)providing a live, intact cell expressing a fusion of a target proteinand a bioluminescent reporter, (b) allowing time for the target proteinto interact with the ligand within the live, intact cell; (c) lysing thecell to produce a cell lysate; (d) contacting the cell lysate with anenvironmentally-sensitive hydrophobic dye, wherein the emission spectraof the bioluminescent reporter and the excitation spectra of theenvironmentally-sensitive hydrophobic dye overlap; (e) treating the celllysate under conditions that cause the target protein to unfold to anappropriate extent; (f) exposing the cell lysate to the substrate of thebioluminescent reporter; (g) measuring signal from theenvironmentally-sensitive hydrophobic dye and bioluminescent reporter inthe cell lysate; and (h) detecting target/ligand interaction based onthe ratio from the signals from said environmentally-sensitivehydrophobic dye and bioluminescent reporter in the cell lysate. In someembodiments, methods further comprise a step of adding the ligand to thelive, intact cell. In some embodiments, the ligand is endogenous to thelive, intact cell. In some embodiments, the signal from saidenvironmentally-sensitive hydrophobic dye in the cell lysate is comparedto a control sample without the ligand.

In some embodiments, provided herein are methods to detect atarget/ligand interaction within a cell, comprising the steps of: (a)providing a live, intact cell expressing a fusion of a target proteinand a bioluminescent reporter, (b) allowing time for the target proteinto interact with the ligand within the live, intact cell; (c) contactingthe live, intact cell with an environmentally-sensitive hydrophobic dye,wherein the emission spectra of the bioluminescent reporter and theexcitation spectra of the environmentally-sensitive hydrophobic dyeoverlap; (d) lysing the cell to produce a cell lysate; (e) treating thecell lysate under conditions that cause the target protein to unfold toan appropriate extent; (f) exposing the cell lysate to the substrate ofthe bioluminescent reporter; (g) measuring signal from theenvironmentally-sensitive hydrophobic dye and bioluminescent reporter inthe cell lysate; and (h) detecting target/ligand interaction based onthe ratio from the signals from said environmentally-sensitivehydrophobic dye and bioluminescent reporter in the cell lysate. In someembodiments, methods further comprise a step of adding the ligand to thelive, intact cell. In some embodiments, the ligand is endogenous to thelive, intact cell. In some embodiments, the signal from saidenvironmentally-sensitive hydrophobic dye in the cell lysate is comparedto a control sample without the ligand.

In some embodiments, provided herein are methods to detect atarget/ligand interaction within a live, intact cell, comprising thesteps of: (a) providing a live, intact cell expressing a fusion of atarget protein and a bioluminescent reporter, (b) allowing time for thetarget protein to interact with the ligand within the live, intact cell;(c) contacting the live, intact cell with an environmentally-sensitivehydrophobic dye, wherein the emission spectra of the bioluminescentreporter and the excitation spectra of the environmentally-sensitivehydrophobic dye overlap; (d) exposing the live, intact cell to thesubstrate of the bioluminescent reporter; (e) measuring signal from theenvironmentally-sensitive hydrophobic dye and bioluminescent reporter inthe live, intact cell under pre-denaturing conditions; (f) treating thelive, intact cell under conditions that cause the target protein tounfold to an appropriate extent; (g) measuring signal from theenvironmentally-sensitive hydrophobic dye and bioluminescent reporter inthe live, intact cell under post-denaturing conditions; and (h)detecting target/ligand interaction based on the difference or ratio insignals from said environmentally-sensitive hydrophobic dye andbioluminescent reporter under the pre-denaturing and post-denaturingconditions in the live, intact cell. In some embodiments, methodsfurther comprise a step of adding the ligand to the live, intact cellprior to step (e). In some embodiments, the ligand is endogenous to thelive, intact cell. In some embodiments, the difference or ratio of thesignal is compared to a difference or ratio of signal in a controlsample without the ligand.

In some embodiments, provided herein are methods to detect target/ligandinteractions within live, intact cells using luminescence-based readout.For example, in some embodiments, provided herein are methods comprisingthe steps of: (a) providing a live, intact cell expressing a fusion of atarget protein and a bioluminescent reporter; (b) allowing time for thetarget protein to interact with the ligand within the live, intact cell;(c) treating the live, intact cell under conditions that cause thetarget protein to unfold to an appropriate extent; (d) exposing thelive, intact cell to the substrate of the bioluminescent reporter, (e)measuring signal from the bioluminescent reporter in the live, intactcell; and (f) detecting target/ligand interaction based on the signalfrom said bioluminescent reporter in the live, intact cell.

In some embodiments, provided herein are methods to detect target/ligandinteractions in live cells using lytic endpoint luminescence-basedreadout. For example, in some embodiments, methods comprise the stepsof: (a) providing a live, intact cell expressing a fusion of a targetprotein and a bioluminescent reporter; (b) allowing time for the targetprotein to interact with the ligand within the live, intact cell; (c)treating the live, intact cell under conditions that cause the targetprotein to unfold to an appropriate extent; (d) lysing the cell toproduce a cell lysate; (e) exposing the cell lysate to the substrate ofthe bioluminescent reporter, (f) measuring signal from thebioluminescent reporter in the lysate; and (g) detecting target/ligandinteraction based on the signal from said bioluminescent reporter in thelysate.

In some embodiments, provided herein are methods to detect target/ligandinteractions within a cell lysate using luminescence-based readout. Forexample, in some embodiments, methods comprise the steps of: (a)providing a live, intact cell expressing a fusion of a target proteinand a bioluminescent reporter; (b) lysing the cell to produce a celllysate; (c) allowing time for the target protein to interact with theligand within the cell lysate; (d) treating the cell lysate underconditions that cause the target protein to unfold to an appropriateextent; (e) exposing the cell lysate to the substrate of thebioluminescent reporter; (f) measuring signal from the bioluminescentreporter in the lysate; and (g) detecting target/ligand interactionbased on the signal from said bioluminescent reporter in the lysate.

In some embodiments, provided herein are methods to detect target/ligandinteractions in live, intact cells using luminescence-based readout withratiometric analysis. For example, in some embodiments, methods comprisethe steps of: (a) providing a live, intact cell expressing a fusion of atarget protein and a bioluminescent reporter, (b) allowing time for thetarget protein to interact with the ligand within the live, intact cell;(c) exposing the live, intact cell to the substrate of thebioluminescent reporter, (d) measuring signal from the bioluminescentreporter in the live, intact cell under pre-denaturing conditions; (e)treating the live, intact cell under conditions that cause the targetprotein to unfold to an appropriate extent; (f) measuring signal fromthe bioluminescent reporter in the live, intact cell underpost-denaturing conditions; and (g) detecting target/ligand interactionbased on the difference or ratio in signal from the bioluminescentreporter under the pre-denaturing and post-denaturing conditions in thelive, intact cell.

In some embodiments, provided herein are methods to detect target/ligandinteractions in a cell lysate using luminescence-based readout withratiometric analysis. For example, in some embodiments, methods comprisethe steps of: (a) providing a live, intact cell expressing a fusion of atarget protein and a bioluminescent reporter; (b) lysing the cell toproduce a cell lysate; (c) allowing time for the target protein tointeract with the ligand within the cell lysate; (d) exposing the celllysate to the substrate of the bioluminescent reporter; (e) measuringsignal from the bioluminescent reporter in the cell lysate underpre-denaturing conditions; (f) treating the cell lysate under conditionsthat cause the target protein to unfold to an appropriate extent; (g)measuring signal from the bioluminescent reporter in the cell lysateunder post-denaturing conditions; and (h) detecting target/ligandinteraction based on the difference or ratio in signal from thebioluminescent reporter under the pre-denaturing and post-denaturingconditions in the cell lysate.

In some embodiments, provided herein are methods to detect target/ligandinteractions in live, intact cells using BRET readout with dye additionfollowing a partial denaturation step (e.g., post-heat). For example, insome embodiments, methods comprise the steps of: (a) providing a live,intact cell expressing a fusion of a target protein and a bioluminescentreporter; (b) allowing time for the target protein to interact with theligand within the live, intact cell; (c) treating the live, intact cellunder conditions that cause the target protein to unfold to anappropriate extent; (d) contacting the live, intact cell with anenvironmentally-sensitive hydrophobic dye, wherein the emission spectraof the bioluminescent reporter and the excitation spectra of theenvironmentally-sensitive hydrophobic dye overlap; (e) exposing thelive, intact cell to the substrate of the bioluminescent reporter; (f)measuring signal from the environmentally-sensitive hydrophobic dye andbioluminescent reporter in the live, intact cell; and (g) detectingtarget/ligand interaction based on the ratiometric signal from saidenvironmentally-sensitive hydrophobic dye and bioluminescent reporter inthe live, intact cell.

In some embodiments, provided herein are methods to detect target/ligandinteractions in live cells using lytic endpoint BRET readout with dyeaddition. For example, in some embodiments, methods comprise the stepsof: (a) providing a live, intact cell expressing a fusion of a targetprotein and a bioluminescent reporter; (b) allowing time for the targetprotein to interact with the ligand within the live, intact cell; (c)treating the live, intact cell under conditions that cause the targetprotein to unfold to an appropriate extent; (d) lysing the cell toproduce a cell lysate; (e) contacting the cell lysate with anenvironmentally-sensitive hydrophobic dye, wherein the emission spectraof the bioluminescent reporter and the excitation spectra of theenvironmentally-sensitive hydrophobic dye overlap; (f) exposing the celllysate to the substrate of the bioluminescent reporter; (g) measuringsignal from the environmentally-sensitive hydrophobic dye andbioluminescent reporter in the cell lysate; and (h) detectingtarget/ligand interaction based on the ratiometric signal from saidenvironmentally-sensitive hydrophobic dye and bioluminescent reporter inthe cell lysate.

In some embodiments, provided herein are methods to detect target/ligandinteractions in cell lysate using BRET readout with dye addition. Forexample, in some embodiments, methods comprise the steps of: (a)providing a live, intact cell expressing a fusion of a target proteinand a bioluminescent reporter; (b) lysing the cell to produce a celllysate; (c) contacting the cell lysate with an environmentally-sensitivehydrophobic dye, wherein the emission spectra of the bioluminescentreporter and the excitation spectra of the environmentally-sensitivehydrophobic dye overlap; (d) allowing time for the target protein tointeract with the ligand within the cell lysate; (e) treating the celllysate under conditions that cause the target protein to unfold to anappropriate extent; (f) exposing the cell lysate to the substrate of thebioluminescent reporter; (g) measuring signal from theenvironmentally-sensitive hydrophobic dye and bioluminescent reporter inthe cell lysate; and (h) detecting target/ligand interaction based onthe ratiometric signal from said environmentally-sensitive hydrophobicdye and bioluminescent reporter in the cell lysate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the effects of heat treatment on HeLa cell viability(FIGS. 1A and 1B) and NANOLUC activity (RLU) within HeLa cells afterexposure to a temperature gradient (FIG. 1C).

FIGS. 2A-B show analysis of melting temperatures of several exampletarget-NANOLUC (Nluc) fusions across a range of target classes asdetermined by NANOLUC activity (RLU) in live, intact cells (FIG. 2A) anddigitonin-treated (FIG. 2B) mammalian cells when exposed to atemperature gradient.

FIGS. 3A-D show a detection of an increase in melting temperature forCDK2-Nluc and Nluc-CDK2 as determined by NANOLUC activity (RLU) afterbinding to a stabilizing ligand in live, intact cells (FIGS. 3A-B) anddigitonin-treated mammalian cells (FIGS. 3C-D) and subsequently exposedto a temperature gradient.

FIG. 4 shows a detection of an increase in melting temperature forCDK2-Nluc as determined by NANOLUC activity (RLU) after binding to astabilizing ligand in live mammalian cells subsequently lysed with lyticNANOGLO Detection Reagent (Promega) and subsequently exposed to atemperature gradient.

FIGS. 5A-F show detection of an increase in melting temperature for apanel of kinases as determined by NANOLUC activity (RLU) after bindingto stabilizing ligands in digitonin treated mammalian cells andsubsequently exposed to a temperature gradient.

FIGS. 6A-D show detection of an increase in melting temperatures or nochange in melting temperatures for CDK2-Nluc and Nluc-ABL1 as determinedby NANOLUC activity (RLU) after incubation with a panel of compoundsthus displaying compound selectivity for the cytoplasmic targets inmammalian cells subsequently exposed to digitonin and a temperaturegradient.

FIG. 7 shows detection of an increase in melting temperatures or nochange in melting temperatures for HDAC1-Nluc as determined by NANOLUCactivity (RLU) after incubation with a panel of compounds thusdisplaying compound selectivity for the nuclear target in mammaliancells and subsequently exposed to digitonin and a temperature gradient.

FIGS. 8A-B show detection of an increase in melting temperature for thetargets KDR-Nluc and DHFR-Nluc as determined by NANOLUC activity (RLU)after incubation with stabilizing ligand in mammalian cells subsequentlyharvested with the compound being washed out and then subjected to atemperature. With this compound washout step, the experiment is analyzednot under compound equilibrium conditions and has the ability to analyzebinding kinetics. KDR is a membrane protein highlighting anothersubcellular location that the assay is able to monitor. This assay wasperformed with live, intact cells throughout the whole assay conditions.

FIGS. 9A-9B show detection of an increase in melting temperature forCDK2-pep86 and pep86-CDK2 as determined by luciferase activity throughspontaneous binary complementation after incubation in the presence of astabilizing ligand in digitonin-treated mammalian cells subsequentlyexposed to a temperature gradient.

FIG. 10 shows detection of an increase in melting temperature forCDK2-NLuc156 as determined by luciferase activity through spontaneousbinary complementation after incubation in the presence of a stabilizingligand in digitonin-treated mammalian cells subsequently exposed to atemperature gradient.

FIG. 11A-11B show temperature and stabilizing ligand concentrationeffects on melting temperature of CDK2-pep86 as determined by luciferaseactivity through spontaneous binary complementation after incubation inthe presence of stabilizing ligand in digitonin-treated mammalian cellssubsequently exposed to a temperature gradient.

FIGS. 12A-B show temperature and stabilizing ligand concentrationeffects on melting temperature of CDK2-11S as determined by luciferaseactivity through spontaneous binary complementation after incubation inthe presence of stabilizing ligand in digitonin-treated mammalian cellssubsequently exposed to a temperature gradient.

FIG. 13 shows detection of ligand binding through a change in meltingtemperature for CDK2-Nluc and LCK-Nluc as determined by bioluminescenceresonance energy transfer (BRET) after incubation in the presence ofstabilizing ligand in digitonin-treated mammalian cells subsequentlyexposed to a temperature gradient.

FIGS. 14A-B show analysis of isothermal dose response curves for a panelof kinase inhibitors with CDK2-Nluc and LCK-Nluc as determined by BRETafter incubation in the presence of different concentrations of theindividual compounds while time of heating and temperature were keptconstant. This highlights the concentration dependence of the thermalstabilization and allows for compound affinity signatures to beobtained.

FIGS. 15A-15B show detection of an increase in melting temperatures(stabilizing ligand) or no change in melting temperatures (non-binding)for cytoplasmic target fusions CDK2-Nluc and Nluc-MAPK14 as determinedby BRET after incubation with a panel of compounds thus displayingcompound selectivity in mammalian cells subsequently exposed todigitonin and a temperature gradient.

FIG. 16 shows detection of an increase in melting temperatures(stabilizing ligand) or no change in melting temperatures (non-binding)for the nuclear target fusion IDAC1-Nluc as determined by BRET afterincubation with a panel of compounds in mammalian cells subsequentlyexposed to digitonin and a temperature gradient.

FIGS. 17A-17C show detection of an increase in melting temperature forCDK2-Nluc as determined by BRET after incubation with stabilizing ligand(staurosporine) in mammalian cells subsequently exposed to digitonin anda temperature gradient using three different environmentally-sensitiveacceptor dyes reporting on protein folding status as BRET acceptor dyes.Dye examples included: Protein Thermal Shift™ Dye (Life Technologies),SYPRO Orange protein gel stain, and SYPRO Red protein gel stain. B_(max)is acceptor dye dose dependent, but that there is no change in theapparent melting temperature (T_(agg)).

FIGS. 18A-18B show detection of an increase in melting temperature forCDK2-Nluc as determined by BRET after incubation with stabilizing ligand(AZD5438) in digitonin-treated mammalian cells subsequently exposed to atemperature gradient and using two different environmentally-sensitivedyes to report on the folded protein status and serve as BRET acceptordyes which was either added before or after the heating step. Dyeexamples included: the dye included in the Life Technologies ThermalShift Assay Dye Kit and the dye included in the Enzo PROTEOSTAT ThermalShift Assay Kit.

FIG. 19 shows detection of an increase in melting temperature forCDK2-Nluc as determined by NANOLUC activity (RLU) and BRET (mBU) afterincubation with stabilizing ligand in mammalian cells subsequentlyexposed to a temperature gradient and either analyzed immediately orincubated at room temperature for 3 minutes prior to analysis.

FIGS. 20A-B show detection of an increase in melting temperature for thenuclear target fusion HDAC1-Nluc as determined by NANOLUC activity (RLU)and BRET (mBU) after incubation with stabilizing ligand in mammaliancells subsequently exposed to a temperature gradient and either analyzedimmediately or incubated at room temperature for 3 minutes prior toanalysis.

FIG. 21 displays prophetic examples of alternative energy transferstrategies combined with environmentally sensitive dyes to use in athermal shift assay.

FIG. 22 is Table 1, providing the order of addition of the componentsfor the experiments depicted in FIGS. 1-20 .

FIGS. 23-34 show graphs depiction characterization of dyes synthesizedin Example 18: left) BRET be used to detect ligand binding through achange in relative BRET compared to DMSO controls (Unt) as exampled withCDK2-Nluc target fusions [+/−Staurosporine (SSP)] in live or lyticconditions [+/−digitonin (Dig)]. As expected, the shape of the BRETcurves are bell-shaped due to the loss of Nluc signal with proteinunfolding and increasing temperatures or dye dissociation upon proteinaggregation or both; middle) the fold change in BRET signal at 56° C.over the background BRET signal at 42° C. for cells transfected withCDK2-Nluc target that were treated with DMSO and varying dyeconcentrations. The larger the signal/background (S/B), the more signalwindow to allow for determining compound binding stability effects. Thisalso allows optimal dye concentrations to be determined; right) the foldchange in BRET signal at 52° C. over the background BRET signal at 42°C. for cells transfected with Nluc-MAPK14 target that were treated withDMSO and varying dye concentrations. The larger the signal/background(S/B), the more signal window to allow for determine compound bindingstability effects, allowing determination of optimal dye concentrations.

FIG. 35 shows fluorescent mode analysis of CS0036.

FIG. 36 shows fluorescent mode analysis of CS0096.

FIG. 37 shows fluorescent mode analysis of CS0048.

DEFINITIONS

As used herein, the terms “fusion,” “fusion protein,” and “fusionpolypeptide” synonymously refer to a chimera of heterologous first andsecond protein or polypeptide segments, for example, a chimera of aprotein of interest (e.g., target protein) joined to a reporter protein(e.g., luciferase). The second protein is typically fused to theN-terminus or C-terminus of the first protein, but may also be insertedinternally within the sequence of the first protein.

As used herein, the term “luciferase” refers to any of a variety ofmonooxygenase enzymes that catalyze the conversion (e.g., oxidation) ofa substrate (e.g., firefly luciferin, latia luciferin, bacterialluciferin, coelenterazine, dinoflagellate luciferin, vargulin, andderivatives thereof) into an excited-energy-state product that emitsenergy in the form of light upon decaying to its ground state.

As used herein, the term “natural polypeptide” as used herein refers toa polypeptide that exists in nature. For example, a “natural luciferase”is a luciferase polypeptide with a sequence and/or any other relevantfeatures (e.g., post-translational modifications) that exists in nature.The term “synthetic polypeptide” refers to a polypeptide having an aminoacid sequence that is distinct from those found in nature (e.g., notnatural). When used in this context, the term “synthetic” does notrelate to the method by which a polypeptide is produced (e.g.,recombinant technology, chemical synthesis, etc.). A “wild-typepolypeptide” or “wild-type luciferase” is the most common variantoccurring in nature; whereas a “variant polypeptide” or “mutantpolypeptide” refers to a polypeptide having an amino acid sequence thatis distinct from the wild-type sequence. A variant or mutant polypeptidemay be natural or synthetic. The aforementioned terms (e.g., “natural”,“synthetic”, “wild-type”, “variant”, and “mutant”) have the samemeanings when used in reference to nucleic acids, genes, peptide,proteins, etc.

As used herein, the term “sequence identity” refers to the degree towhich two polymer sequences (e.g., peptide, polypeptide, nucleic acid,etc.) have the same sequential composition of monomer subunits. The term“sequence similarity” refers to the degree with which two polymersequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similarpolymer sequences. For example, similar amino acids are those that sharethe same biophysical characteristics and can be grouped into thefamilies, e.g., acidic (e.g., aspartate, glutamate), basic (e.g.,lysine, arginine, histidine), non-polar (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan) anduncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). The “percent sequence identity” (or “percentsequence similarity”) is calculated by: (1) comparing two optimallyaligned sequences over a window of comparison (e.g., the length of thelonger sequence, the length of the shorter sequence, a specified window,etc.), (2) determining the number of positions containing identical (orsimilar) monomers (e.g., same amino acids occurs in both sequences,similar amino acid occurs in both sequences) to yield the number ofmatched positions, (3) dividing the number of matched positions by thetotal number of positions in the comparison window (e.g., the length ofthe longer sequence, the length of the shorter sequence, a specifiedwindow), and (4) multiplying the result by 100 to yield the percentsequence identity or percent sequence similarity. For example, ifpeptides A and B are both 20 amino acids in length and have identicalamino acids at all but 1 position, then peptide A and peptide B have 95%sequence identity. If the amino acids at the non-identical positionshared the same biophysical characteristics (e.g., both were acidic),then peptide A and peptide B would have 100% sequence similarity. Asanother example, if peptide C is 20 amino acids in length and peptide Dis 15 amino acids in length, and 14 out of 15 amino acids in peptide Dare identical to those of a portion of peptide C, then peptides C and Dhave 70% sequence identity, but peptide D has 93.3% sequence identity toan optimal comparison window of peptide C. For the purpose ofcalculating “percent sequence identity” (or “percent sequencesimilarity”) herein, any gaps in aligned sequences are treated asmismatches at that position.

As used herein, the term “sample” is used broadly to refer to any ofbiological samples (e.g., fluids, tissues, etc.) and environmentalsamples as well as reaction mixtures or other multicomponent solutionsand mixtures.

As used herein, the term “complex sample” refers to a sample comprisinga large number and variety of different compounds, polymers,macromolecules, complexes, etc. A complex sample may comprise buffers,salts, peptides, polypeptides, proteins (including also enzymes),carbohydrates (complex and simple carbohydrates), lipids, fatty acids,fat, nucleic acids, organelles and other cellular components, etc.Examples of complex samples include cells, e.g., live intact cells, celllysates, body fluids (e.g., blood (or blood products), saliva, urine,etc.), tissues (e.g., biopsy tissue), cells grown in vitro andsubsequently injected into animal in vivo and recollected for ex vivoanalysis, cells in 3D culture, cells in tissues, reaction mixtures, etc.In particular embodiments, a complex samples contain a target protein aswell as additional non-target peptides, polypeptides, and/or proteins.

As used herein, the term “quenched” refers to a decrease in fluorescenceemission from a fluorescent entity (e.g., dye) upon interaction of aparticular substance (e.g., water) or condition, relative to thefluorescence emission from the fluorescent entity when not interactingwith the particular substance or condition. The term “quenched” itselfdoes not place any limitation on the extent of the decrease influorescence. The degree of quenching may be expressed at a percentageof fluorescence in the quench state compared to the unquenched state(e.g., 10 RLU in the quenched state compared to 100 RLU in theunquenched state is 90% quenching).

As used herein, the term “BRET” is used to describe the occurrence ofbioluminescence resonance energy transfer between a bioluminescent donor(e.g., a luciferase protein) and an acceptor fluorophore. It is adistance-dependent interaction in which energy is transferred from thedonor bioluminescent protein and substrate to an acceptor fluorophorewithout emission of a photon. The efficiency of BRET is dependent on theinverse sixth power of the intermolecular separation, making it usefulover distances comparable with the dimensions of biologicalmacromolecules (e.g., within 30-80 Å, depending on the degree ofspectral overlap).

As used herein, the term “ligand for a target protein” refers to amolecular entity that binds to a target protein. The ligand may be asmall molecule, peptide, antibodies, macromolecules (e.g., nucleic acid,viral proteins, bacterial proteins, polysaccharides, syntheticpolymers), or other molecular entity that bind the target protein. Theterm “test ligand” refers to a molecular entity that is being assayedfor the capacity to bind the target protein.

DETAILED DESCRIPTION

Provided herein are systems and methods for characterizing target/ligandengagement. In particular, luciferase-labeled polypeptide targets areused to detect or quantify target/ligand engagement (e.g., within a cellor cell lysate).

In some embodiments, compositions (e.g., fusions of target polypeptidesand luciferase reporter), systems (e.g., kits or reaction mixturescomprising, for example, test ligands, luciferase substrates, assayreagents, cells, e.g., live intact cells, cell lysates, etc.) andmethods are provided for monitoring (e.g., detecting, quantitating,etc.) target/ligand engagement in complex (cellular) environments (e.g.,based on the biophysical principle of ligand-induced thermalstabilization of target proteins). In some embodiments, the luminescentsignal generated by a fusion of a target protein and luciferase (e.g.,NANOLUC (Promega Corp., Madison, Wis.); see, e.g., U.S. Pat. Nos.8,557,970 and 8,669,103, both of which are herein incorporated byreference in their entireties) or a luciferase peptide/polypeptideinteracts with a complement polypeptide/peptide to produce an activeenzyme (e.g., NLPep and NLPoly (Promega Corp., Madison, Wis.); see,e.g., U.S. Pub. No. 2014/0348747, which is herein incorporated byreference in its entirety) is monitored over temperature rangesufficient to result in unfolding and aggregation of the unbound targetprotein, both in the presence and absence of a ligand or test ligand forthe target protein. An altered or shifted luminescent signal over therange of temperatures indicates interaction between the target proteinand ligand. In some embodiments, the luminescent signal (e.g., relativelight units (RLU)) from the luciferase reporter portion of the fusion isdirectly monitored. In some embodiments, bioluminescence resonanceenergy transfer (BRET) from the luciferase reporter portion of thefusion to an environmentally sensitive dye (e.g., a dye that interactswith the hydrophobic portions of the target protein as they becomeexposed at higher temperatures, a protein-aggregation detection dye,etc.) is measured.

In some embodiments, the target/luciferase fusion is expressed in cells(e.g., a live intact cell or a relevant cell line), and the cells and/ora cell lysate thereof, is exposed to a ligand of interest (e.g., ligandfor the target protein, test ligand (e.g., potential drug), etc.) andsubsequently exposed to conditions (e.g., increased temperature) capableof causing the unfolding and/or aggregation of the unbound targetprotein. In such embodiments, a target protein that has been stabilizedby interaction with ligand in the cell or cell lysate will exhibit analtered or shifted melting temperature as detected by alteration of theluminescent or BRET signal in the presence and absence of ligand.

In some embodiments in which luciferase signal is directly detected as ameasure of thermal shift, luciferase substrate (and purifiedcomplementary peptide/polypeptide in the case complementation assays(e.g., using NLPep and NLPoly (Promega Corp., Madison, Wis.); see, e.g.,U.S. Pub. No. 2014/0348747, which is herein incorporated by reference inits entirety)) is added to the samples (e.g., cells, cell lysate, etc.)for analysis. In other embodiments in which BRET signal from theluciferase of the fusion to an environmentally sensitive dye (e.g., adye that interacts with the hydrophobic portions of the target proteinas they become exposed at higher temperatures, a protein-aggregationdetection dye, etc.) is detected as a measure of thermal shift,luciferase substrate (and purified complementary peptide/polypeptide inthe case complementation assays (e.g., using NLPep and NLPoly)) and theenvironmentally sensitive dye are added to the samples (e.g., cells,cell lysate, etc.) for analysis. In some embodiments, detergents (e.g.Digitonin), lysis buffer (e.g. NANOGLO Lytic reagent), and/or otherreagents are added to the sample. In some embodiments, luminescenceand/or BRET is analyzed using, for example, by a luminometer at one ormore specific temperatures (e.g., encompassing a temperature in whichthe target protein unfolds). Shift of the signal (e.g., luciferasesignal or BRET signal) in the presence vs. absence of ligand indicatesinteraction (e.g., binding) of the target and ligand.

It has long been recognized that the binding of low molecular weightligands increases the thermal stability of a protein (Koshland (1958).Proc Natl Acad Sci USA. 44 (2): 98-104.; Linderstrøm-Lang & Schellman(1959). The Enzymes. 1(2) 443-510.; herein incorporated by reference inits entirety). A TSA detects this stabilizing effect by measuring thethermal stability of a target protein in the presence and absence of aligand, thereby detecting or quantifying the target/ligand interaction.A traditional TSA is a fluorescence-based method for monitoringtarget/ligand interactions (WO 1997/020952; herein incorporated byreference in its entirety); the primary weakness of such an assay isthat it detects the unfolding of any proteins present in the samplebeing assayed (e.g., target and non-target protein), and therefore canonly be used to analyze purified protein. Because target/ligandinteractions may be affected by a multitude of factors in vivo, atraditional TSA of purified protein may be of limited informationalvalue. Other TSA-type assays have been developed (See, e.g., Moreau M J,et al. Quantitative determination of protein stability and ligandbinding using a green fluorescent reporter system. Mol. Biosyst. (6);1285-1292. 2010; and CETSA, U.S. Pub No. 2014/0057368; hereinincorporated by reference in their entireties); however, these assaysrely on post-denaturation quantification of soluble vs. denaturedprotein as a measure of target protein stability and require multiplepurification and/or detection steps. Provided herein are assays that canbe used to identify ligands that bind to a target protein and/or toquantify the affinity of such interactions. The assays described hereinare distinct from other methods (e.g., traditional thermal shift, GFPreporter systems, CESTA, etc.), for example, because: (1) the assaysallow characterization of target/ligand interactions with non-purifiedprotein; (2) the assays can be conducted in a cell, cell lysate, orother complex liquid containing many different biomolecules; (3) theassay technology does not rely upon antibodies recognizing targetprotein epitopes; (4) the assays are homogeneous; (5) the assays usecommon and simple reagents, materials and instruments (e.g., aluminometer); and/or (6) assays are not limited to a single read.

In some embodiments, the systems and methods described herein utilizefusion constructs (e.g., fusion polypeptides and nucleic acids andvectors encoding them) comprising a target protein (e.g., the bindingcharacteristics of which are being examined) and a reporter peptide orpolypeptide. In some embodiments, any protein of interest may find useas the target protein. Assays described herein provide for analysis ofthe binding characteristics (e.g., potential ligands, binding affinity,etc.) of such proteins. A reporter of the fusion construct may be anypeptide or polypeptide, the activity of which can be detected within acell, e.g., a live intact cell, or cell lysate, in real-time. While thescope of embodiments herein is not limited by the identity of thereporter, many embodiments utilize a luciferase reporter. In suchembodiments, a suitable luciferase is fused (e.g., directly or by alinker (e.g., peptide or other linker moiety)) to the N-terminus orC-terminus of a target protein (or inserted internally within the targetprotein) to generate a fusion polypeptide for use in the systems andmethods herein. In some embodiments, nucleic acid constructs areprovided that encode fusion polypeptides (e.g., N-target-reporter-C,N-reporter-target-C, N-target-linker-reporter-C,N-reporter-linker-target-C, etc.). In some embodiments, vectors (e.g.,plasmids, bacmids, cosmids, viral vectors (e.g., lentivirus vectors,adeno-associated viral vectors (AAVs), etc.), etc.) comprising nucleicacid constructs that encode fusion polypeptides (e.g., along withappropriate incorporation and/or expression elements) are provided. Insome embodiments, cells (e.g., bacterial, mammalian, human, etc.)transformed or transfected (e.g., transiently or stably) with nucleicacids and/or vectors encoding fusion polypeptides useful for assaysdescribed herein are provided.

Although the reporter of a fusion construct may be one that exhibits anysuitably detectable activity, in some embodiments, the reporter is aluciferase enzyme. Suitable luciferase enzymes include those selectedfrom the group consisting of: Photuris pyralis or North American fireflyluciferase; Luciola cruciata or Japanese firefly or Genji-botaruluciferase; Luciola italic or Italian firefly luciferase; Luciolalateralis or Japanese firefly or Heike luciferase; Luciola mingrelica orEast European firefly luciferase; Photuris pennsylvanica or Pennsylvaniafirefly luciferase; Pyrophorus plagiophthalamus or Click beetleluciferase; Phrixothrix hirtus or Railroad worm luciferase; Renillareniformis or wild-type Renilla luciferase; Renilla reniformis Rluc8mutant Renilla luciferase; Renilla reniformis Green Renilla luciferase;Gaussia princeps wild-type Gaussia luciferase; Gaussia princepsGaussia-Dura luciferase; Cypridina noctiluca or Cypridina luciferase;Cypridina hilgendorfii or Cypridina or Vargula luciferase; Metridialonga or Metridia luciferase; and Oplophorus luciferase (e.g.,Oplophorus gracilirostris (OgLuc luciferase), Oplophorus grimaldii,Oplophorus spinicauda, Oplophorus foliaceus, Oplophorus noraezeelandiae,Oplophorus typus, Oplophorus noraezelandiae or Oplophorus spinous). Forany of the above luciferases, fusions may contain a wild-type ornaturally-occurring variant of the luciferase or may comprise asynthetic version (e.g., optimized for one or more characteristics(e.g., emission, stability, etc.). In some embodiments, assays arecarried out both in the presence and absence of a ligand for the targetprotein (or a test ligand) and in the presence of the appropriatesubstrate for the luciferase. A negative control assay may be performedin the absence of substrate. A positive control may be performed withthe un-fused luciferase and appropriate substrate in the presence andabsence of ligand and/or in the presence an absence of un-fused targetprotein.

In some embodiments, a substrate for the reporter (e.g., bioluminescentreporter (e.g., luciferase, etc.), etc.) is provided. In someembodiments, a bioluminescent reporter converts the substrate into areaction product and releases light energy, e.g., luminescence, as abyproduct. In some embodiments, the substrate is a substrate for aluciferase enzyme. Appropriate substrates for known reporters areunderstood in the field.

In some embodiments, a fusion construct comprises a target protein fusedto a Oplophorus grachlorostris luciferase (OgLuc). The fusion maycomprise a natural (e.g., wild-type or variant) OgLuc sequence or maycomprise a synthetic OgLuc (e.g., optimized for one or morecharacteristics (e.g., luminescence, signal stability, proteinstability, etc.), etc.). The natural wild-type OgLuc sequence is givenin SEQ ID NO: 1. Some suitable Oplophorus luciferases are described, forexample, in U.S. Pat. Nos. 8,669,103 and 8,557,970. In some embodiments,a luciferase polypeptide comprises at least 60%(e.g., >65%, >70/6, >75%. >80%, >85%, >90° %, >95%, >98%, >99%, 100%/,and any ranges therein) sequence identify with SEQ ID NO: 1. In someembodiments, comprises a amino acid substitutions at positions relativeto one or more of positions: 1, 2, 4, 6, 10, 11, 14, 15, 16, 18, 19, 20,21, 22, 23, 24, 25, 27, 28, 31, 32, 33, 34, 36, 38, 39, 40, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 54, 55, 56, 58, 59, 60, 66, 67, 68, 69, 70,71, 72, 74, 75, 76, 77, 86, 87, 89, 90, 92, 93, 94, 95, 96, 97, 98, 99,100, 102, 104, 106, 109, 110, 111, 112, 113, 115, 117, 119, 124, 125,126, 127, 128, 129, 130, 135, 136, 138, 139, 142, 143, 144, 145, 146,147, 148, 149, 150, 152, 154, 155, 159, 158, 163, 166, 167, 168, or 169of SEQ ID NO: 1. In some embodiments, a luciferase exhibits one or moreof enhanced luminescence, enhanced signal stability, and enhancedprotein stability relative to a wild-type Oplophorus luciferase. In someembodiments, comprises at least 60%(e.g., >65%, >70%, >75%. >80%, >85%, >90%, >95%, >98%, >99%, 100%, andany ranges therein) sequence identify with SEQ ID NO: 2. In someembodiments, comprises SEQ ID NO: 2.

In some embodiments, a target protein is fused to a first peptide orpolypeptide (e.g., that does not independently exhibit substantialdetectable activity) that forms an active reporter construct throughstructural complementation with a second polypeptide or peptide (See,e.g. U.S. Ser. No. 14/209,610 and U.S. Ser. No. 14/209,546; hereinincorporated by reference in their entireties.). In such embodiments,only one of the two (or more) elements (peptide or polypeptide) thatform the reporter construct is fused to the target. The structuralcomplement of the fused element of the reporter construct is addedseparately to the system (e.g., cell (e.g., added exogenously, expressedby the cell), cell lysate, in vitro system, etc. In particularembodiments, a target protein is fused to a first peptide or polypeptide(e.g., that doesn't independently exhibit a detectable activity) thatforms an active luciferase construct through structural complementationwith a second polypeptide or peptide (See, e.g. U.S. Ser. No. 14/209,610and U.S. Ser. No. 14/209,546; herein incorporated by reference in theirentireties.). In such embodiments, the fusion polypeptide will notindependently catalyze a significant amount of substrate ahigh-energy-state product that will produce light upon return to astable state. Rather, only in the presence of the complement polypeptideor peptide is the active luciferase construct formed. Embodiments willtypically be described as comprising a target fused to a completereporter; however, unless indicated otherwise, it should be understoodthat the reporter may be formed by structural complementation ofmultiple (e.g., 2, 3, or more) elements, only one of which is fused tothe target.

In some embodiments, a first peptide (e.g., fused to the target protein)comprises at least 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, and anyranges therein) but less than 100% sequence identify with SEQ ID NO: 3,and a complement polypeptide comprises at least 60% (e.g., 65%, 70%,75%, 80%, 85%, 90%, 95%, and any ranges therein) but less than 100%sequence identify with SEQ ID NO: 4. In some embodiments, the firstpeptide comprises an amino acid substitutions at positions relative toone or more of positions: G157del, T159S, C164F, E165K, N166K, L168S,A169del of SEQ ID NO: 3 (wherein SEQ ID NO: 3 numbering is 157-169). Insome embodiments, the complement polypeptide comprises a amino acidsubstitutions at positions relative to one or more of positions: Q11E,G15A, F31L, G35A, L46R, G51A, G67A, G71A, M75E, 176V, H93P, I107L,D108N, N144T, L149M, 157S (addition of S at 157 position) of SEQ ID NO:4 (wherein SEQ ID NO: 4 numbering is 1-156).

In some embodiments, a first polypeptide (e.g., fused to the targetprotein) comprises at least 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%,95%, and any ranges therein) but less than 100% sequence identify withSEQ ID NO: 4, and a complement polypeptide comprises at least 60% (e.g.,65%, 70%, 75%, 80%, 85%, 90%, and any ranges therein) but less than 100%sequence identify with SEQ ID NO: 3. In some embodiments, the firstpolypeptide comprises a amino acid substitutions at positions relativeto one or more of positions (e.g., Q11E, G15A, F31L, G35A, L46R, G51A,G67A, G71A, M75E, I76V, H93P, 1107L, D108N, N144T, L149M, 157S (additionof S at 157 position) of SEQ ID NO: 4 (wherein SEQ ID NO: 4 numbering is1-156)). In some embodiments, the complement peptide comprises a aminoacid substitutions at positions relative to one or more of positions(e.g., G157del, T159S, C164F, E165K, N166K, L168S, A169del of SEQ ID NO:3 (wherein SEQ ID NO: 3 numbering is 157-169)).

Depending upon the identity of the bioluminescent reporter used, anappropriate substrate will be selected, for example, from thoseincluding, but not limited to: firefly luciferin, latia luciferin,bacterial luciferin, coelenterazine, dinoflagellate luciferin, vargulin,and suitable derivatives thereof. In some embodiments, the substrate isa substrate for an Oplophorus luciferase, e.g., NANOLUC enzyme fromPromega Corporation (e.g., SEQ ID NO: 2). In some embodiments, thesubstrate comprises coelenterazine, a coelenterazine derivative, astructural or functional equivalent of coelenterazine, a moleculesubstantially equivalent to coelenterazine (e.g., structurally and/orfunctionally), or molecule functionally or structurally similar tocoelenterazine. In some embodiments, the bioluminescent reporterconverts the coelenterazine, coelenterazine derivative, structural orfunctional equivalent of coelenterazine, or substantial equivalent tocoelenterazine into coelenteramide, a coelenteramide derivative, astructural or functional equivalent of coelenteramide, or a substantialequivalent to coelenteramide and releases light energy as a byproduct.

In some embodiments, a reporter is an epitope tag (See FIG. 21 , toppanel). In such embodiments, a labeled (e.g., fluorescently labeled)antibody that recognizes and binds the epitope is included. In someembodiments, BRET occurs between the label on the antibody and thefluorescent dye when both are bound to the epitope-tagged targetprotein. In some embodiments, the epitope is accessible to the antibodywhen the protein is folded or unfolded. In other embodiments, somedegree of target unfolding is required for the antibody to access theepitope tag. In some embodiments, following a thermal denaturation step,addition of a detection antibody labeled with a donor fluorophore (e.g.terbium, europium, etc.) is used in a FRET assay withdenaturation/aggregation-sensitive (e.g., environmentally-sensitive dye,hydrophobic dye, etc.) dye as a FRET acceptor. Ligand-mediated thermalstabilization results in a loss of FRET/TR-FRET signal and/or therequirement of more denaturing conditions (e.g., higher temperature) toachieve the FRET/TR-FRET signal.

In some embodiments, two epitope tags (e.g. FLAG-V5) are fused to atarget protein (See FIG. 21 , bottom panel). In such embodiments, theepitopes are accessible to separate, differently-labeled antibodies(e.g., donor and acceptor labeled) for the respective epitopes when thetarget protein is folded (FRET signal is produced). Upon partial orcomplete unfolding of the target, the epitopes become unavailable andthe FRET signal is lost.

In some embodiments, the formation of a fusion protein does notsubstantially impact or alter the stability or function of either thetarget or reporter relative to the function of each individual protein.For example, for a fusion of target enzyme and a luciferase, theformation of the fusion does not substantially alter the stability ofeither portion, the luminescence of the luciferase, the signal stabilityof the luciferase, or the activity of the enzyme.

In some embodiments, the target polypeptide and reporter polypeptide(e.g., luciferase) are connected directly without intervening amino acidsequence. For example, the target polypeptide may be fused to theN-terminus or the C-terminus of the reporter. In other embodiments, thetarget polypeptide and reporter polypeptide (e.g., luciferase) areconnected by a linker moiety or connector sequence. In some embodiments,a linker provides connection and allows a desired amount ofspace/distance between the elements (e.g., to reduce interactionsbetween target and reporter). Although a linker is typically a peptidelinker, in some embodiments, other chains or polymers may be utilized.In some embodiments, a connector sequence comprises or consists of oneor more amino acids (e.g., a peptide or polypeptide chain of 2-50 aminoacids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and any rangestherein). In some embodiments, the presence of a connector sequence in afusion protein does not substantially impact or alter the stability orfunction of either the target or reporter relative to the function ofeach individual protein. For example, for a fusion of target enzyme anda luciferase, the presence of a connector sequence does notsubstantially alter the stability of either portion, the light output ofthe luciferase, or the activity of the enzyme. In some embodiments, aconnector sequence is included in a fusion to prevent interactionsbetween the target and reporter that could affect the stability ofactivity of either or both portions. For any particular combination ofproteins in a fusion, a wide variety of connector sequences may beemployed. In one embodiment, the connector sequence is a sequencerecognized by an enzyme, e.g., a cleavable sequence. For instance, theconnector sequence may be one recognized by a caspase or TEV protease,or may be a chemically-cleavable or photocleavable sequence.

In some embodiments, the activity (e.g., luminescence) of a reporter(e.g., luciferase) fused to a target protein is directly detected. Insome embodiments, the activity (e.g., luminescence) of a reporterconstruct (e.g., luciferase dimer), a portion of which is fused to atarget protein (e.g., the structural complement portion is not fused tothe target protein), is directly detected. In some embodiments, reporteractivity is measured at one or more temperatures as a measure of targetprotein stability. In some embodiments, at lower temperatures (e.g.,<50° C., <48° C., <46° C., <44° C., <42° C., <40° C., <38° C., <36° C.,34° C., <32° C., <30° C., and ranges therein (e.g., 30-50° C., 35-45°C., etc.), depending upon the target and reporter stabilities) both thetarget and reporter are stably folded. As temperatures are increased,and one or both of the target and reporter begin unfolding oraggregating, signal from the reporter decreases (e.g., the activity ofthe reporter decreases as it becomes unfolded or aggregated). In someembodiments, because the reporter and target are fused, a more stablethe target protein results in a more stable reporter and reportersignal. Similarly, a less stable target results in destabilization ofthe reporter and a loss, or reduction, of reporter signal at a lowertemperature. In some embodiments, if a ligand binds to the targetprotein, the target protein is stabilized. Since, in some embodiments,stabilization of the target protein results in stabilization of thereporter and enhancement of signal stability, binding of the ligand tothe target can be observed as an increase in the stability of thereporter signal. Embodiments in which the signal from the reporter(e.g., luciferase) at a given temperature (e.g., around the T. of theprotein) or temperature range is directly used as a measure of proteinstability are referred to herein as “direct detection.”

In some embodiments, ligand binding destabilized the target protein. Insome embodiments, the reporter is thermally stable or labile compared tothe protein of interest it is fused to.

In some embodiments, the activity (e.g., luminescence) from atarget/reporter fusion is measured at one or more temperatures (e.g.,over a range of temperatures (e.g., from below the T_(m) of the targetprotein to above the T_(m) of the target protein)), both in the presenceand absence of a ligand for the target or a test ligand. If the ligandor test ligand binds to or interacts with the target protein under theconditions assayed (e.g., in a cell, in a cell lysate, etc.), acorresponding shift in the activity of the reporter is observed. Forexample, at a given temperature (e.g., near the T. of the targetprotein) the activity (e.g., luminescence (e.g., RLUs)) of the reporter(e.g., luciferase) is increased. If the unfolding of the protein isanalyzed at a range of temperatures, so as to determine a T_(m), ligandbinding and stabilization of the target protein is apparent as a shift(e.g., right shift) in the T_(m) to a higher temperature.

Traditional thermal shift assays utilize purified target protein and adye that (1) binds nonspecifically to hydrophobic surfaces and (2) isquenched by water (e.g., Sypro Orange). In such an assay, at lowertemperatures (e.g., <50° C., <48° C., <46° C., <44° C., <42° C., <40°C., <38° C., <36° C., 34° C., <32° C., <30° C., and ranges therein(e.g., 30-50° C., 35-45° C., etc.), depending upon the target stability)the target is stably folded, and the hydrophobic regions of the targetare buried and inaccessible to the hydrophobic dye. Under these foldedconditions, the dye cannot significantly bind to the target protein, andtherefore fluorescence from the dye is quenched by water in the aqueousenvironment. As temperatures are increased, the target begins unfoldingand/or aggregating, the hydrophobic regions of the target becomereveled, and the dye binds to the target. Upon binding the targetprotein, the dye becomes unquenched and the fluorescent signalincreases. The more stable the target, the higher the temperaturerequired to observe the fluorescence increase. Similarly, a less stabletarget results in destabilization of the target and an increase influorescent signal at a lower temperature. In such an assay, if a ligandbinds to the target protein, the target protein is stabilized, which canbe observed as an increase in the temperature required to generate thefluorescent signal.

The traditional TSA relies on nonspecific binding of the dye (e.g.,Sypro Orange) to the hydrophobic regions of the target protein.Therefore, if proteins other than the target protein are present in theassay, the dye will bind to those proteins as well (e.g., as they becomeunfolded), thereby confounding the results. For at least this reason,traditional TSAs are performed using purified target protein and cannotbe performed in cells, cell lysate, or other complex environments.

The direct detection embodiments described above overcome the limitationof the traditional TSA by detecting activity of a reporter fused to thetarget protein. The unfolding of proteins not fused to the reporter(e.g., proteins present in the cellular milieu or cell lysate) do notdirectly affect the signal. In another embodiment, a BRET TSA isprovided that makes use of the target-specificity of thedirect-detection embodiments described herein (e.g., only the unfoldingof proteins fused to a reporter are monitored (e.g., even in a complexenvironment)), as well as the sensitivity of the traditional TSA (e.g.,the increase in fluorescence from the dye is directly correlated withthe degree of unfolding of the target protein). In such embodiments, afusion of a target protein and reporter (e.g., luciferase) is generated,and fluorescence measurements are taken in the presence of anenvironmentally-sensitive dye (e.g., a dye that: (1) bindsnonspecifically to hydrophobic surfaces, (2) has an excitation spectrathat significantly overlaps the emission spectra of the reporter, andoptionally (3) the fluorescence of which is quenched by water). Theactivity of the dye may be quenched by water (e.g., as in a traditionalTSA), or the dye may be active (e.g., capable of emitting light at aparticular wavelength in response to an excitation) whether bound to aprotein or free in solution. In some embodiments, because the dye willbe excited via BRET from the reporter and not directly, the capacity forthe dye to fluoresce when not bound to the target (e.g., when bound toother proteins within a cell or cell lysate.) does not affect the assay(e.g., because only target-bound dye is within the range (e.g., 1-10 nm)to receive energy transfer from the reporter). In some embodiments, atlower temperatures (e.g., <50° C., <48° C., <46° C., <44° C., <42° C.,<40° C., <38° C., <36° C., 34° C., <32° C., <30° C., and ranges therein(e.g., 30-50° C., 35-45° C., etc.), depending upon the target stability)the target is stably folded, and the hydrophobic regions of the targetare buried and inaccessible to the hydrophobic dye. Under these foldedconditions, signal from the reporter (e.g., fused to the target) isdetected, but because the dye cannot significantly bind to the foldedtarget protein (e.g., because hydrophobic regions are no accessible onthe folded target) fluorescence from the dye as a result of BRET is notdetected (e.g., significant direct reporter signal is observed, but BRETto the dye is not observed, is decreased, and/or is minimal). Theproximity limitation of BRET prevents significant energy transfer fromoccurring between the reporter and unbound dye. In embodiments in whichthe dye is quenched by water, unbound dye is also not detected by directexcitation (e.g., from a light source). In embodiments in which the dyemaintains its activity in water, while not producing a signal from BRET,the unbound dye may be detected by direct excitation (e.g., from a lightsource). As temperatures are increased, the target begins unfoldingand/or aggregating, the hydrophobic regions of the target becomerevealed, the dye binds to the target, and the dye becomes unquenched(e.g., the quantum yield of the dye increases). Under thesepartially-folded or unfolded conditions, BRET occurs from the reporterto the bound dye (e.g., dye bound to the revealed hydrophobic portionsof the target protein), and fluorescence from the dye, as the result ofBRET, is detectable as a direct measure of (e.g., is proportional to)target protein unfolding. The more stable the target, the higher thetemperature required to observe the increase in BRET. Similarly, a lessstable target results in destabilization of the target and an increasein the BRET signal at a lower temperature. In such an assay, if a ligandbinds to the target protein, the target protein is stabilized, which canbe observed as an increase in the temperature required to generate theBRET signal (e.g., to generate increased BRET signal (e.g., increasedover background)). In BRET TSA, although the hydrophobic dye will bindto any proteins present in the complex sample (e.g., cell, cell lysate,etc.), only the target protein is fused to the reporter, therefore theBRET signal will be specific to the target protein. Due to the proximityconstraints placed on BRET, significant energy transfer only occursbetween the reporter and target-bound dye. The BRET signal is madepossible by 1) presence of reporter (e.g., NLuc) activity and 2) bindingof the dye to the fusion protein allowing the reporter to donate andexcite the dye to emit at a certain wavelength. The fusion of thereporter to the target provides the target specificity; the dye providesa readout of the target protein state (e.g., folded, partially folded,unfolded, etc.).

In some embodiments, BRET TSAs described herein utilize fluorogenicdyes. In other embodiments, dyes are non-fluorogenic.

In some embodiments, BRET TSAs described herein utilize fluorescent dyesthat: (1) are quenched by an aqueous environment (e.g., when not boundby a target); (2) interact with (e.g., bind) hydrophobic surfaces, sucha hydrophobic peptide segments; and (3) exhibit enhanced or increasedfluorescence upon binding a hydrophobic surface.

Certain fluorescent compounds exhibit only a weak fluorescence emissionwhen free in aqueous solution (Semisotnov, et al., Biopolymers 31:119,1991; herein incorporated by reference in its entirety), but fluorescemuch more strongly when bound to organized hydrophobic surfaces. Bindingof these compounds to fully folded globular proteins is typically weak,since hydrophobic residues are predominantly buried in the interior ofthe protein. Furthermore, binding of these compounds to random coilconformations (as found in fully unfolded or denatured polypeptides) isalso disfavored because in these conformations hydrophobic residues,though exposed, are not sufficiently well organized to support highaffinity binding of the probes. The dyes, however, typically bind withhigher affinity and stoichiometry to compact unfolded proteinconformations, such as “molten globules”, which are characterized bycompactness relative to random coil unfolded states, the presence ofsubstantial secondary structure, and the lack of a unique overallconformation. These probes may be referred to herein as“environmentally-sensitive hydrophobic dyes.”

In some embodiments, BRET TSAs described herein utilize fluorescent dyesthat bind to the hydrophobic regions of unfolding proteins, moltenglobules, and aggregates. Due to the proximity limitations of BRET,significant energy transfer from the bioluminescent report to thefluorescent dye only occurs when the dye is bound to the unfoldingtarget protein. When the target protein is fully folded, the fluorescentdye does not bind the target protein, the dye is not within theproximity of the bioluminescent reporter, and BRET does not occur. Insuch embodiments, the bioluminescent reporter provides the system'sspecificity for the target protein while the fluorescent dye providesthe measure of protein unfolding.

Examples of dyes that find use in embodiments described herein includeSYPRO Orange, 1-anilino-8-naphthalene sulfonate (ANS),bis-1-anilino-8-naphthalene sulfonate (bis-ANS),6-propionyl-2-(N,N-dimethyl)-aminonaphthalene (Prodan) (MolecularProbes, Eugene, Oreg.), SYPRO Red, SYPRO Ruby, SYPRO Tangerine, and NileRed. Other suitable dyes are those listed in Table 1.

TABLE 1 Optical Property (in DMSO) Structure MS Emission ExcitationReference #

487.2 468-490 620 CS0000

375.6 468 618 CS0004

431.7 468-490 622 CS0007

543.9 468-490 620 CS0008

487.7 468 602 CS0020

377.3 450 526 CS0013

477.3 430-450 580 CS0010

427.2 405 505 CS0017

399.5 489-514 644 CS0018

513.9 468 620 CS0043

537.9 560 680 CS0028

513.6 516 702 CS0024

359.7 470-480 620 CS0075

479.7 (M⁺) 480 624 CS0101

451.7 (M⁺) 480 624 CS0036

711.1 (M⁺) 480 622 CS0100

654.9 (M⁺) 480 622 CS0045

776.1 478 620 CS0048

695.1 480 620 CS0073

455.7 (M⁺) 550 604 CS0067

553.9 550 604 CS0068

499.8 (M⁺) 480 618 CS0096

537.9 532 640 CS0081

429.8 (M⁺) 532 640 CS0085

404.6 400 550 CS0086

296.4 (M⁺) 400 550 CS0087

365.6 468 620 CS0117

711.8 (M⁺) 480 620 CS0121

393.6 (M⁺) 480 620 CS0112

406.3 (M²⁺) 480 620 CS0158

491.8 (M²⁺) 516 702 CS0155

737.2 (M⁺) 516 702 CS0147

537.8 462 654 CS0038

537.8 not fluorescent CS0071

537.8 not fluoresent CS0072

523.7 CS000X

In some embodiments, combinations of dyes are used. In some embodiments,a combination of dyes having varying binding properties is used, suchthat the dye mix finds use with target proteins having differentstructural characteristics. Other dyes (e.g., dyes known in the field)may also find use in embodiments described herein.

One advantage of some embodiments described herein is the ability of theassays to be performed in complex samples (e.g., those containingnon-target proteins). In some embodiments, samples that find use withthe TSAs described herein include cells (e.g., cell culture, cell samplefrom a patient, etc.), tissue samples (e.g., tissue from biopsy), celllysates (e.g., lysed of cells expressing a target/reporter fusion, celllysate expressing target/reporter fusion in vitro, cell lysate withexogenous target/reporter fusion, etc.), complex reaction mixtures(e.g., in vitro reaction mixtures comprising the components necessaryfor the assay as well as non-essential components (e.g., non-targetproteins, competitor proteins, competitor ligands, etc.), simple invitro reaction mixtures comprising the components necessary for theassay, etc.), etc.

In some embodiments, reporter and/or BRET signal is detected underconditions that favor protein folding and/or conditions that favor somedegree of protein unfolding. Two means of varying conditions aretemperature and protein denaturants. For example, lower temperatures(e.g., <50° C., <48° C., <46° C., <44° C., <42° C., <40° C., <38° C.,<36° C., 34° C., <32° C., <30° C., and ranges therein (e.g., 30-50° C.,35-45° C., etc.)) favor protein folding, while higher temperatures(e.g., >50° C., >55° C., >60° C., >65° C., >70° C., >75° C., >80°C., >85° C., >90° C., >95° C., and ranges therein (e.g., 60-90, ° C.55-75° C., etc.)) favor protein unfolding, exposure of hydrophobicregions, and/or aggregation. Similarly, the presence of variousdenaturants and increasing concentrations thereof, favor proteinunfolding. Numerous protein denaturants are known in the art and couldbe empirically selected for use herein. Without being limited to aparticular protein denaturant, exemplary protein denaturants includeguanidinium thiocyanate, guanidinium hydrochloride, arginine, sodiumdodecyl sulfate (SDS), urea, or any combination thereof. In someembodiments, a combination of one or more protein denaturants andincreased temperature is employed.

In some embodiments, the reporter signal and/or BRET signal (e.g., theresult of energy transfer from bioluminescent protein toenvironmentally-sensitive hydrophobic dye) is detected under a singlecondition, and a comparison is made between the signals in the presenceand absence of ligand. In such embodiments, the signal is typicallydetected under partially denaturing conditions, or conditions in whichthe unbound target protein is beginning to unfold, but it is expectedthat the ligand-bound target will not be unfolding or will be lessunfolded (as will be apparent from the change in signal (e.g., directreporter signal or BRET signal, depending upon the assay)). Appropriateconditions for such a single-point assay can be determined empiricallybased on the stability of the particular target protein. In someembodiments, conditions (e.g., temperature and/or denaturantconcentration) are selected at or near (e.g., ±5%, ±10%, ±15%, ±20%,±25%, ±30%, ±35%, ±40%, ±45%, ±50%) the melting temperature (T_(m)) forthe target protein. The melting temperature (e.g., in the presence ofthe intended denaturant concentration) of the target protein can bedetermined using any of a variety of well-known techniques, including,but not limited to: UV/visible spectrometry, nuclear magnetic resonancespectroscopy (NMR), circular dichroism (CD), etc.

In other embodiments, the reporter signal and/or BRET signal (e.g., theresult of energy transfer from bioluminescent protein toenvironmentally-sensitive hydrophobic dye) is detected under multipleconditions or over a range of conditions (e.g., varying temperatureand/or denaturant concentration), and a comparison is made between thesignals in the presence and absence of ligand. In such embodiments,signal may be detected at a first condition in which both theligand-bound and inbound target are expected to be well folded. Undersuch conditions, similar signals are typically (although not always)expected in both the presence and absence of ligand. In someembodiments, signal is also detected under one or more additionalconditions (e.g., increasing temperature, increasing pressure, and/orincreasing denaturant concentration) approaching and/or exceeding theexpected T_(m) (e.g., under the denaturant concentration) of the targetprotein in the absence of ligand. As increasingly unfavorable foldingconditions are applied, a difference will be observed between the signal(e.g., from a direct reporter signal or BRET signal, depending upon theassay) from a ligand-bound sample and an unbound target sample.

The number of conditions to assay may be made by weighing factors suchas: desired speed of the assay, desired precision/accuracy of themeasurements, affinity of binding to be detected, etc. For example, insome embodiments, an assay detects signal under a first condition inwhich the target is well-folded, a second condition near the T_(m) ofthe unbound target, and a third condition in which the unbound target isexpected to be unfolded and/or aggregated. These conditions may bedetermined empirically using the methods described herein or othertechniques for monitoring protein folding that are known in the field.As another example, an assay makes stepwise (e.g., every 0.1° C., every0.2° C., every 0.5° C., every 1.0° C., every 2.0° C., every 5.0° C.,etc.) signal detections (e.g., from reporter and/or BRET) over a rangeof temperatures (e.g., from: 10° C., 15° C., 20° C., 25° C., 30° C., 35°C., 40° C., etc.; to: 50° C., 55° C., 60° C., 65° C., 70° C., 75° C.,80° C., 85° C., 90° C., 95° C., etc.), and or a range of denaturantconcentrations.

In some embodiments, a melt-curve analysis is performed for each sample,according to the detected signals at the various conditions assayed. Insome embodiments, a visual or automated analysis of the melt-curves inthe presence and absence of ligand reveals target/ligand interaction orbinding. In some embodiments, a melting temperature (T_(m)) iscalculated for each sample, according to the detected signals at thevarious conditions assayed. In some embodiments, a comparison of T_(m)'sin the presence and absence of ligand reveals target/ligand interactionor binding.

In some embodiments, assays described herein are performed in cells,e.g., live intact cells, or in a lysate of cells. In such embodiments, anucleic acid construct encoding a target/reporter fusion may betransfected or transformed into cells in order to allow expression ofthe fusion within cells (e.g., live intact cells, cells in which theassay will be performed, cells which will be lysed before the assay isperformed, etc.). In some embodiments, assays described herein providethe detection of molecular interactions between a target and ligandwithin a cell, e.g., a live intact cell, or in living organisms (e.g.,bacteria, yeast, eukaryotes, mammals, primates, human, etc.). In someembodiments, a target/reporter fusion protein is expressed in the cell,e.g., a live intact cell, or whole organism, and signal is detectedunder various conditions in the presence and absence of ligand. In someembodiments, cells are transiently and/or stably transformed ortransfected with vector(s) (e.g., encoding target/reporter fusionprotein, structural complement (e.g., NLPep and NLPoly), etc.). In someembodiments, transgenic organisms are generated that code for thenecessary assay components (e.g., target/reporter fusion protein,structural complement (e.g., NLPep and NLPoly), etc.) for carrying outthe assays described herein. In other embodiments, vectors are injectedinto whole organisms. A suitable vector may be viral (e.g., lentivirus,AAV, etc.) or non-viral (e.g., plasmid, bacmid, etc.). In someembodiments, in addition to encoding a target/reporter fusion, a vectorfurther comprises elements to allow/promote expression of as much.

In some embodiments, other assay components not expressed in cells ororganisms (e.g., reporter substrate, ligand, environmentally-sensitivehydrophobic dye, etc.) are added to the cell or organism exogenously. Inembodiments in which assays are not performed in vivo, thetarget/reporter fusion may also be added exogenously.

Embodiments described herein provide compositions, systems, and methodsthat are useful in a variety of fields including basic research, medicalresearch, drug discovery, etc. The reagents and assays described hereinare not limited to any particular applications, and any usefulapplication should be viewed as being within the scope of the presentinvention

Typical applications that make use of embodiments described hereininvolve the monitoring/detection of protein-ligand interactions. Suchassays are useful for monitoring molecular interactions under anysuitable conditions (e.g., in vitro, in vivo, in situ, whole animal,etc.), and find use in, for example, drug discovery, elucidatingmolecular pathways, studying equilibrium or kinetic aspects of complexassembly, high throughput screening, etc.

In some embodiments, assays are used to elucidate the affinity of, orunderstand the interaction of, a protein of interest and a potentiallyassociated entity of interest (protein, nucleic acid, small molecule,etc.) under complex conditions (e.g., intracellularly).

Embodiments described herein find use in drug screening and/or drugdevelopment. For example, the interaction of a small molecule drug or anentire library of small molecules with target protein of interest (e.g.,therapeutic target) is monitored (e.g., within aphysiologically-relevant cell line, etc.). In some embodiments, drugscreening applications are carried out in a high through-put format toallow for the detection of the binding of tens of thousands of differentmolecules to a target, or to test the effect of those molecules on thebinding of a ligand.

In some embodiments, provided herein is the detection of target/ligandinteractions in living organisms (e.g., bacteria, yeast, eukaryotes,mammals, primates, human, etc.) and/or cells. In some embodiments, cellsare transiently and/or stably transformed or transfected with vector(s)(e.g., encoding target/reporter fusions, complement polypeptide, etc.).In some embodiments, transgenic organisms are generated that code forthe necessary components (e.g., encoding target/reporter fusions,complement polypeptide, etc.) to carry out the assays described herein.In other embodiments, vectors are injected into whole organisms.

EXPERIMENTAL Example 1 Materials and Methods

Method for Expression Plasmid Construction for Full LengthNANOLUC-Target Fusions (Applies to Examples Using Full Length NANOLUC)

To produce NANOLUC fusions with protein targets of interest, pF31K Nuc[CMV/Neo] and pF32 [CMV/Neo] were used to place NANOLUC at theN-terminus or the C-terminus of the target protein (respectively) usingthe manufacturer's protocol (Promega). cDNAs encoding the followingtarget proteins were made and were a 100% match to their respective NCBIreference sequence identifiers; CDK2, DHFR, KDR, SRC, EZH2, HDAC1, BRD4,MAPK14, LCK, ABL, DDR1, and BTK.

Method for Expression Plasmid Construction for NLPep and NLPoly NLPepand NLPoly Target Fusions (pep86-CDK2, CDK2-pep86, CDK2-Nluc156,CDK2-pep11S)

For plasmid constructions, the source of CDK2 was pFN21-CDK2 (PromegaFLEXI vector backbone) received from Kazusa DNA Research Institute(Chiba, Japan).

Pep86-CDK2: This construct was built by transferring CDK2 to the FLEXIvector, pF-n5K3-86-GSSG-barnase, according to Promega's recommendedprotocols in the FLEXI vector technical manual.

CDK2-Pep86: This construct was built by transferring CDK2 to the FLEXIvector, pF-c5K3-barnase-GSSG-86, according to Promega's recommendedprotocols in the FLEXI vector technical manual.

CDK2-Nluc156: This construct was built by first transferring CDK2 to theFLEXI vector, pF-nCA-bamase-GSSG-Nuc, according to Promega's recommendedprotocols in the FLEXI vector technical manual. The Nuc portion of thisvector was then modified by site-directed mutagenesis to contain a G51Asubstitution. It was then further modified by site-directed mutagenesisto contain a deletion of the sequence coding for the final 13 aminoacids of Nluc.

CDK2-11S: This construct was built by transferring CDK2 to the FLEXIvector, pF-c5K3-barnase-GSSG-11S, according to Promega's recommendedprotocols in the FLEXI vector technical manual.

Each of the constructs contained a GSSG linker between CDK2 and eitherNLPep86, Nluc156, or NLpoly11S.

Method for Generation of Purified Detection NLPep86 and NLPoly11S

NLPep86 (SEQ ID NO: 5) (containing an N-terminal acetyl and C-terminalamide) was made synthetically by Peptide 2.0. NLpoly1S (SEQ ID NO:6)(NLpoly11S with C-terminal 6His tag) was isolated by transforming E.coli KRX cells (Promega) with the FLEXI vector, pF6HisCK, and thenoverexpressing and purifying according to Promega's recommended protocolfor isolating His-tagged proteins (HISLINK Protein Purificationtechnical manual).

General Protocol Cell Transfection for all Experiments:

NANOLUC-target or NLPep and NLPoly-target fusion constructs were dilutedinto carrier DNA (pGEM3ZF-, Promega) at a mass ratio of 1:10 or 1:100(mass/mas) prior to forming FuGENE HD complexes according to themanufacturer's protocol (Promega). DNA:FuGENE complexes were formed at aratio of 1:3 (ug DNA/uL FuGENE), then 1 part of the transfectioncomplexes was then mixed with 20 parts (volume/volume) of HeLa cells(ATCC) suspended at a density of 2×10⁵ in DMEM (Gibco)+10% FBS(Hyclone), followed by incubation in a humidified, 37° C./5% CO₂incubator for 18-24 hours.

Example 2 Direct-Luciferase-Detection Thermal Shift Assay with FullLength NANOLUC

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal from a luciferasefused to the target protein.

To ensure the NANOLUC (Nluc) luminescent signal was originating fromintact cells after the cells had been exposed to a temperature gradient,the integrity of the cells and cell membranes after heating wasinvestigated as well as measuring Nluc activity in the cells versus inthe supernatant. HeLa cells were heated to different temperatures,cooled at room temperature, mixed with trypan blue, and the absolutecell numbers and dye exclusion were measured. Parallel samples were spundown to pellet, and the cells and supernatant collected.

HeLa cells were transfected with CDK2-Nluc DNA at a 1:10 DNA ratio andincubated overnight. The cells were trypsinized using Trypsin-EDTA(0.25%) (Gibco) and re-suspended in Opti-MEM without phenol red(Gibco)+1× Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 100 uL/well volume, and the plates were placed into a thermal cycler(MW Research) where the samples were heated individually at differenttemperatures spanning a gradient of 42-66° C. across the plate for 3minutes followed by cooling at 22° C. for 3 minutes. To determine theabsolute cell count and viability, 10 uL cell aliquot from eachtemperature was mixed with an equal volume of 0.4% (w/v) trypan blueexclusion dye solution (Life technologies) and analyzed using theCountess Automated cell counter (Life Technologies). To determine wherethe Nuc luminescent signal was originating, the 96-well PCR plate wascentrifuged to pellet the cells. The supernatant and cell pellet wascollected separately and placed into a 96-well tissue culture plate(Corning). Furimazine Live Cell Substrate (Promega) was added to a finalconcentration of 1×, and luminescence was measured on a BMG Clariostarluminometer equipped with a 450 nm BP filter. Samples were alsocollected from a 96-well PCR plate that had not undergone centrifugationand placed into a tissue culture plate followed by addition offurimazine substrate and luminescence analysis.

The data demonstrates that the cells were not lysed, even at highertemperatures, since the number of cells remained unchanged and membranesremained intact as determined by dye exclusion (FIG. 1A-B). Furthermore,the Nluc activity was retained in the cells with the luciferase signaloriginating from the cell samples and not the supernatant post-heattreatment (FIG. 1C).

Example 3 Direct-Luciferase-Detection Thermal Shift Assay with FullLength NANOLUC

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal from a luciferasefused to the target protein.

HeLa cells were transfected separately with the various Nluc-fusion DNAat a 1:10 DNA ratio and incubated overnight. The cells were trypsinizedusing Trypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM withoutphenol red (Gibco)+1× Protease Inhibitor Cocktail (Promega) which hadbeen reconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 90 uL/well volume, and either treated with 10 uL/well of a 10% DMSOsolution (Sigma) or 10 uL/well of a 0.5 mg/ml solution of digitonin(Sigma). The plates were placed into a thermal cycler (MW Research)where the samples were heated individually at different temperaturesspanning a gradient of 42-66° C. across the plate for 3 minutes followedby cooling at 22° C. for 3 minutes. 90 uL/well samples were thentransferred to a tissue culture plate (Corning), and Furimazine LiveCell Substrate (Promega) was added to a final concentration of 1×.Luminescence was measured on a BMG Clariostar luminometer equipped witha 450 nm BP filter. Data was normalized by relating RLU signals to theRLU of the lowest temperature for the respective sample. Data was thenfitted to obtain apparent melting temperature where the protein isprecipitating (Ta values) using the Boltzmann Sigmoidal equation withGraphpad Prism software.

Protein thermal melt curves can be generated for Nluc-target fusionsspanning several different target classes and subcellular localization(nuclear, cytoplasmic, and membrane) in live, intact cells (FIG. 2A),and cells that had been treated with the detergent digitonin (FIG. 2B)with results indicating each protein displayed distinct melting curvesas analyzed by luciferase activity and reported as apparent aggregationtemperatures (T_(agg)).

Example 4 Direct-Luciferase-Detection Thermal Shift Assay with FullLength NANOLUC

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal from a luciferasefused to the target protein.

To determine if a shift in T_(agg) after exposure to stabilizing ligandcan be detected, the thermal melt curve for CDK2, which showed adistinct melt curve in the presence of DMSO (control), was evaluated.When AZD5438 (a drug known to bind to CDK2) was added, clear shifts inthe melting curves with increases in apparent T_(agg) were detectedcompared to DMSO controls and analyzed by luciferase activity indicatingspecific binding and protein stabilization of the CDK2 by the inhibitor.

HeLa cells were transfected with CDK2-Nluc or Nluc-CDK2 DNA at a 1:10DNA ratio and incubated overnight. The cells were trypsinized usingTrypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM without phenolred (Gibco)+1× Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 90 uL/well volume and treated with 10 uL of 10% DMSO (Sigma), 10uL/well of a 0.5 mg/ml solution of digitonin (Sigma), 10 uL of 1 mMAZD5438 (Selleckchem) in 10% DMSO, and/or 10 uL of 1 mM Methotrexate(Sigma) in 10% DMSO. The plates were then incubated for 1-2 hours in ahumidified, 37° C./5% CO₂ incubator. The plates were placed into athermal cycler (MW Research) where the samples were heated individuallyat different temperatures spanning a gradient of 42-66° C. across theplate for 3 minutes followed by cooling at 22° C. for 3 minutes. 90uL/well samples were then transferred to a tissue culture plate(Corning), and Furimazine Live Cell Substrate (to a final concentrationof 1×; Promega) or NANOGLO lytic reagent (Promega) was added.Luminescence was measured on a BMG Clariostar luminometer equipped witha 450 nm BP filter. Data was normalized by relating RLU signals to theRLU signals of the lowest temperature for the respective sample. Datawas then fitted to obtain apparent melting temperature where the proteinis precipitating (T_(agg) values) using the Boltzmann Sigmoidal equationwith Graphpad Prism software.

The thermal shift induced by AZD5438 was insensitive to Nluc positioningon the N- or C-terminal of CDK2 (FIG. 3 ) and was demonstrated in live(FIG. 3A-B) and digitonin-treated cells (FIG. 3C-D) as well as in cellslysed with the addition of lytic substrate (FIG. 4 ).

Example 5 Direct-Luciferase-Detection Thermal Shift Assay with FullLength NANOLUC

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal from a luciferasefused to the target protein.

To demonstrate that the assay can be applied to a larger class of kinaseproteins, specific melt curves and thermal shifts across a panel ofkinases were obtained after exposure to specific stabilizing compounds.HeLa cells, expressing different kinase Nluc-target fusions with varyingfunctions and subcellular localization, were exposed to compounds knownto target each one and then exposed to the temperature treatment.

HeLa cells were transfected separately with the various Nluc-target DNAat a 1:10 DNA ratio and incubated overnight. The cells were trypsinizedusing Trypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM withoutphenol red (Gibco)+1× Protease Inhibitor Cocktail (Promega) which hadbeen reconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 80 uL/well volume and treated with 10 uL/well of a 0.5 mg/ml solutionof digitonin (Sigma), 10 uL of 1 mM AZD5438 (Selleckchem) in 10% DMSO,10 uL of 0.25 mM AMG548 (Tocris) in 10% DMSO, 10 uL of 1 mM Dasatinib(BioVision) in 10% DMSO, or 10 uL of 0.5 mM Staurosporine (LCLaboratories) in 10% DMSO. The plates were then incubated for 1 hour ina humidified, 37° C./5% CO₂ incubator. The plates were placed into athermal cycler (MW Research) where the samples were heated individuallyat different temperatures spanning a gradient of 42-66° C. across theplate for 3 minutes followed by cooling at 22° C. for 3 minutes.Furimazine Live Cell Substrate (Promega) was added to a finalconcentration of 1× in 20 uL/well volume. 100 uL/well was thentransferred to a tissue culture plate (Corning), and luminescence wasmeasured on a BMG Clariostar luminometer equipped with a 450 nm BPfilter. Data was normalized by relating RLU signals to the RLU signalsof the lowest temperature for the respective sample. Data was thenfitted to obtain apparent melting temperature where the protein isprecipitating (T_(agg) values) using the Boltzmann Sigmoidal equationwith Graphpad Prism software.

Obvious shifts in melting temperature, with increases in T_(agg)≥4° C.in all cases, were seen when the cells were treated with the specificbinding compounds as compared to DMSO controls for each kinase tested(FIGS. 5A-F).

Example 6 Direct-Luciferase-Detection Thermal Shift Assay with FullLength NANOLUC

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal from a luciferasefused to the target protein.

To demonstrate the assay's ability to determine compound targetselectivity and highlight the assay's potential screening capabilitiesfor proposed inhibitors, an assay against two kinase target fusions,CDK2-Nluc and Nuc-ABL1, with the same panel of kinase inhibitors knownto have different selectivity against these two kinase targets wasperformed. The assay accurately validated ligand selectivity, and rankorder potency, with the compounds known to bind to CDK2 and/or ABL1causing a shift in melting temperature (apparent Tau), and thosecompounds known not to have affinity towards CDK2 and/or ABL1 leavingthe melting temperature relatively unchanged, as compared to the DMSOcontrol.

HeLa cells were transfected separately with CDK2-Nluc or Nluc-ABL 1 DNAat a 1:100 DNA ratio and incubated overnight. The cells were trypsinizedusing Trypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM withoutphenol red (Gibco)+1× Protease Inhibitor Cocktail (Promega) which hadbeen reconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 90 uL/well volume and treated with 10 uL of 10% DMSO (Sigma) or 10 uLof a 1 mM stock solution of test compound in 10% h DMSO. Test compoundsincluded: Staurosporine (LC Laboratories), Dasatinib (BioVision),Nilotinib (BioVision), Ponatinib (SYNKinase), AMG548 (Tocris), SB203580(AdipoGen), and AZD5438 (Selleckchem). The plates were then incubatedfor ½ hour in a humidified, 37° C./5% CO₂ incubator prior to addition of10 uL/well of a 0.5 mg/ml solution of digitonin (Sigma) and 10% DMSO(Sigma). The plates were placed into a thermal cycler (MW Research)where the samples were heated individually at different temperaturesspanning a gradient of 42-66° C. across the plate for 3 minutes followedby cooling at 22° C. for 3 minutes. Furimazine Live Cell Substrate(Promega) was added to a final concentration of 1× in 20 uL/well volume.100 uL/well was then transferred to a tissue culture plate (Corning),and luminescence was measured on a BMG Clariostar luminometer equippedwith a 450 nm BP filter. Data was normalized by relating RLU signals tothe RLU of the lowest temperature for the respective sample. Data wasthen fitted to obtain apparent melting temperature where the protein wasprecipitating (Ta values) using the Boltzmann Sigmoidal equation withGraphpad Prism software.

For the CDK2 target fusion, the known target inhibitors, staurosporine,Ponatinib, and AZD5438, all caused a significant increase in apparentT_(agg). Dasatinib, Nilotinib, AMG548, and SB20358, which are allcompounds not known to interact with CDK2, did not result in asignificant shift in apparent T_(agg) compared to DMSO controls whenincubated with cells expressing the CDK2-Nluc fusion (FIG. 6A-B).Similarly, Staurosporine, Dasatinib, Nilotinib, and Ponatinib, all ofwhich target ABL1 kinase, caused a significant shift in apparent T_(agg)compared to DMSO controls, whereas the negative ligands AMG548,SB203580, and AZD5438 did not as expected (FIG. 6C-D).

Example 7 Direct-Luciferase-Detection Thermal Shift Assay with FullLength NANOLUC

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal from a luciferasefused to the target protein.

To demonstrate the TSA approach described herein can be applied broadly,the experiment performed in FIGS. 6A-D was repeated using a targetprotein that maintains a nuclear subcellular localization and originatedfrom a drug target class very different from kinases. Cells weretransfected with HDAC1-Nluc fusions and tested against a panel of knownHDAC inhibitors with Staurosporine, a kinase inhibitor, serving as anadditional negative control with DMSO.

HeLa cells were transfected with HDAC1-Nluc DNA at a 1:100 DNA ratio andincubated overnight. The cells were trypsinized using Trypsin-EDTA(0.25%) (Gibco) and re-suspended in Opti-MEM without phenol red(Gibco)+1× Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 90 uL/well volume and treated with 10 uL of 10% DMSO (Sigma) or 10 uLof a 1 mM stock solution of test compound in 10% DMSO. Test compoundsincluded: SAHA (TOCRIS), Mocetinostat (Selleckchem), Panibinostat (LCLaboratories), ACY1215 (Selleckchem), and Staurosporine (LCLaboratories). The plates were then incubated for ½ hour in ahumidified, 37° C./5% CO₂ incubator prior to addition of 10 uL/well of a0.5 mg/ml solution of digitonin (Sigma) and Furimazine Live CellSubstrate (final concentration 1× (Promega)). The plates were placedinto a thermal cycler (MW Research) where the samples were heatedindividually at different temperatures spanning a gradient of 42-66° C.across the plate for 3 minutes followed by cooling at 22° C. for 3minutes. 100 uL/well was then transferred to a tissue culture plate(Corning), and luminescence was measured on a BMG Clariostar luminometerequipped with a 450 nm BP filter. Data was normalized by relating RLUsignals to the RLU of the lowest temperature for the respective sample.Data was then fitted to obtain apparent melting temperature where theprotein is precipitating (T_(agg) values) using the Boltzmann Sigmoidalequation with Graphpad Prism software.

All four HDAC known inhibitors (SAHA, Mocetinostat, Panibinostat, andACY1215) produced a significant shift in melting temperature (apparentT_(agg)) indicating ligand binding compared to the controls as analyzedby luciferase activity (FIG. 7 ).

Example 8

Direct-luciferase-detection thermal shift assay with full length NANOLUCExperiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal from a luciferasefused to the target protein.

To determine if the assay can be performed when the ligands added arenot maintained under equilibrium conditions, the assay was performedwith a compound washout step. This would also confirm that the targetengagement was happening in live cells as well as have the ability toanalyze binding kinetics.

HeLa cells were transfected with KDR-Nluc or DHFR-Nluc at a 1:10 DNAratio and incubated overnight in t75 tissue culture flasks (Corning).Media was aspirated and replaced with media containing a finalconcentration of 100 uM BIBF-1120 (Selleckchem) for cells expressingKDR-Nluc or 100 uM Methotrexate (Sigma) for cells expressing DHFR-Nluc.DMSO at a final concentration of 1% was used as control and added toeach cell population. The cells were allowed to incubate with compoundsfor 2 hours in a humidified, 37° C./5% CO₂ incubator. The cells weretrypsinized using Trypsin-EDTA (0.25%) (Gibco) and re-suspended inOpti-MEM without phenol red (Gibco)+1× Protease Inhibitor Cocktail(Promega) which had been reconstituted in DMSO (Sigma). Cells wereseeded into 96-well tall-chimney PCR plate (Fisherbrand) at a density of2×10⁴ cells/well in a 100 uL/well and placed into a thermal cycler (MWResearch) where the samples were heated individually at differenttemperatures spanning a gradient of 42-66° C. across the plate for 3minutes followed by cooling at 22° C. for 3 minutes. 90 uL/well was thentransferred to a tissue culture plate (Corning), and Furimazine LiveCell Substrate (final concentration 1× (Promega)) was added.Luminescence was measured on a BMG Clariostar luminometer equipped witha 450 nm BP filter. Data was normalized by relating RLU signals to theRLU of the lowest temperature for the respective sample. Data was thenfitted to obtain apparent melting temperature where the protein isprecipitating (T_(agg) values) using the Boltzmann Sigmoidal equationwith Graphpad Prism software.

FIGS. 8A-B show detection of an increase in melting temperature for thetargets KDR-Nuc (FIG. 8A) and DHFR-Nluc (FIG. 8B) as determined byNANOLUC activity (RLU) after incubation with known stabilizing ligand inmammalian cells subsequently harvested with the compound being washedout, and then subjected to a temperature. KDR is a membrane proteinhighlighting another subcellular location that the assay is able tomonitor.

Example 9 Direct-Luciferase-Detection Thermal Shift Assay with NLPep andNLPoly Through Spontaneous Binary Complementation

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal in which theluminescent signal results through spontaneous binary complementationfrom a peptide fused to the target protein with the complementarysubunit present in the detection reagent.

To determine if the luciferase-based thermal shift assay describedherein could be performed using a spontaneous binary complementation,the NLPep and NLPoly technology (Promega; See, e.g., U.S. Pub. No.2014/0348747: herein incorporated by reference in its entirety) wasused.

HeLa cells were transfected with CDK2-NLpep86 or NLpep86-CDK2 DNA at a1:10 DNA ratio and incubated overnight. The cells were trypsinized usingTrypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM without phenolred (Gibco)+Ix Protease Inhibitor Cocktail (Promega), which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 90 uL/well volume and treated with 1 uL of 100% DMSO (Sigma) or 1 uLof a 10 mM stock solution of AZD5438 (Selleckchem) in 10/a DMSO, and 10uL/well of a 0.5 mg/ml solution of digitonin (Sigma). The plates werethen incubated for 1-2 hour in a humidified, 37° C./5% CO₂ incubator.The plates were placed into a thermal cycler (MW Research) where thesamples were heated individually at different temperatures spanning agradient of 42-66° C. across the plate for 3 minutes followed by coolingat 22° C. for 3 minutes. 90 uL/well was then transferred to a tissueculture plate (Corning), and the complementary subunit, NLpoly11S, wasadded to a final concentration of 1 uM with Furimazine Live CellSubstrate (final concentration 1× (Promega)).

Luminescence was measured on a BMG Clariostar luminometer equipped witha 450 nm BP filter. Data was normalized by relating RLU signals to theRLU of the lowest temperature for the respective sample and subtractingthe background luminescence signal generated by NLpoly11S. Data was thenfitted to obtain apparent melting temperature where the protein isprecipitating (T_(agg) values) using the Boltzmann Sigmoidal equationwith Graphpad Prism software.

FIG. 9 shows detection of an increase in melting temperature forCDK2-NLpep86 and NLpep86-CDK2 as determined by luciferase activitythrough spontaneous binary complementation after incubation in thepresence of the stabilizing ligand AZD5438 in digitonin-treatedmammalian cells subsequently exposed to a temperature gradient andaddition of substrate and complementary subunit NLpoly11S as compared tothe DMSO control.

Example 10 Direct-Luciferase-Detection Thermal Shift Assay with NLPepand NLPoly Through Spontaneous Binary Complementation

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal in which theluminescent signal results through spontaneous binary complementationfrom a peptide fused to the target protein with the complementarysubunit present in the detection reagent.

The following experiment is similar to Example 9, but used another pairof complementary subunits, NLpoly156 (SEQ ID NO: 7) and NLpep86.NLpoly156 was fused to the target protein, and NLpep86 was placed in thedetection reagent.

HeLa cells were transfected with CDK2-Nluc156 DNA at a 1:10 DNA ratioand incubated overnight. The cells were trypsinized using Trypsin-EDTA(0.25%) (Gibco) and re-suspended in Opti-MEM without phenol red(Gibco)+1× Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 90 uL/well volume and treated with 1 uL of 100% DMSO (Sigma) or 1 uLof a 10 mM stock solution of AZD5438 (Selleckchem) in 10% h DMSO, and 10uL/well of a 0.5 mg/ml solution of digitonin (Sigma). The plates werethen incubated for 1-2 hour in a humidified, 37° C./5% CO₂ incubator.The plates were placed into a thermal cycler (MW Research) where thesamples were heated individually at different temperatures spanning agradient of 42-66° C. across the plate for 3 minutes followed by coolingat 22° C. for 3 minutes. 90 uL/well was then transferred to a tissueculture plate (Corning), and the complementary subunit, NLpep86, wasadded to a final concentration of 1 uM with Furimazine Live CellSubstrate (final concentration 1× (Promega)). Luminescence was measuredon a BMG Clariostar luminometer equipped with a 450 nm BP filter. Datawas normalized by relating RLU signals to the RLU of the lowesttemperature for the respective. Data was then fitted to obtain apparentmelting temperature where the protein is precipitating (Tau values)using the Boltzmann Sigmoidal equation with Graphpad Prism software.

FIGS. 10A-C demonstrates the detection of an increase in meltingtemperature for CDK2-NLpoly156 as determined by luciferase activitythrough spontaneous binary complementation after incubation in thepresence of the stabilizing ligand AZD5438 in digitonin-treatedmammalian cells subsequently exposed to a temperature gradient andaddition of substrate and complementary subunit NLpep86 as compared tothe DMSO control.

Example 11 Direct-Luciferase-Detection Thermal Shift Assay with NLPepand NLPoly Through Spontaneous Binary Complementation

Experiments were conducted during development of embodiments of thepresent invention to demonstrate direct detection of the stabilizationof a target protein by the interaction with a target ligand viatemperature dependent alteration of luminescent signal in which theluminescent signal results through spontaneous binary complementationfrom a peptide fused to the target protein with the complementarysubunit present in the detection reagent.

HeLa cells were transfected with CDK2-NLpep86 or CDK2-NLpoly11S DNA at a1:10 DNA ratio and incubated overnight. The cells were trypsinized usingTrypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM without phenolred (Gibco)+Ix Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 90 uL/well volume and treated with 1 uL of a 100× stock solution in adilution series of AZD5438 (Selleckchem) in 10/DMSO, and 10 uL/well of a0.5 mg/ml solution of digitonin (Sigma). The plates were then incubatedfor 1-2 hour in a humidified, 37° C./5% CO₂ incubator. The plates wereplaced into a thermal cycler (MW Research) where the samples were heatedindividually at different temperatures spanning a gradient of 42-66° C.across the plate for 3 minutes followed by cooling at 22° C. for 3minutes. 90 uL/well was then transferred to a tissue culture plate(Corning) and complementary subunit NLpoly1 S or NLpep86 was added to afinal concentration of 1 uM with Furimazine Live Cell Substrate (finalconcentration 1× (Promega)). Luminescence was measured on a BMGClariostar luminometer equipped with a 450 nm BP filter. For EC₅₀analysis, compound concentration was plotted against RLU, and the datawas transformed prior to being fitted using the sigmoidal dose response(variable slope) equation with Graphpad Prism software. For apparentT_(agg) analysis, data was normalized by relating RLU signals to the RLUof the lowest temperature for the respective. Data was then fitted toobtain apparent melting temperature where the protein is precipitating(T_(agg) values) using the Boltzmann Sigmoidal equation with GraphpadPrism software.

FIGS. 11 and 12 demonstrates the temperature and stabilizing ligandconcentration dependency of the luciferase-based thermal shift assaywhich is consistent with other reported methods of thermal shift assay.Here, the EC₅₀ of the ligand AZD5438 shifts as a function of temperatureand the apparent T_(agg) shifts as a function of stabilizing ligandconcentration. In this case, the targets consisted of CDK2-NLpep86fusions (FIGS. 11A-C) and CDK2-NLpoly11S fusions (FIGS. 12A-B).

Example 12 BRET-Detection Thermal Shift Assay

Experiments were conducted during development of embodiments of thepresent invention to demonstrate detection of the stabilization of atarget protein by the interaction with a target ligand via temperaturedependent alteration of BRET signal generated by the energy transferfrom a luciferase fused to the target protein to an environmentallysensitive dye that binds to all proteins unfolding in the sampleincluding the target protein.

Protein thermal melting curves can be generated for purified proteins inwhich the extent of unfolding is measured by the gain in fluorescentsignal from environmentally sensitive dyes that bind to the exposedhydrophobic surfaces of the protein as they unfold. Taking advantage ofthis property and adding these dyes to the luciferase-based thermalshift assay described herein, the environmentally-sensitive dye binds tothe target protein-Nuc fusion, and BRET is produced upon addition ofsubstrate when there is active Nluc available to excite the dye. The dyeacts as a fluorescent BRET acceptor, and the transfer of energy ismoderated by the proximity of the two partners. While the dye will bindto all proteins unfolding in the sample, the luciferase fusion gives thetarget protein specificity; hence any BRET signal is a direct readout onthe state of the protein of interest.

HeLa cells were transfected with CDK2-Nluc or Nluc-LCK DNA at a 1:100DNA ratio and incubated overnight. The cells were trypsinized usingTrypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM without phenolred (Gibco)+1× Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well inan 80 uL/well volume and treated with 10 uL/well of a 0.5 mg/ml solutionof digitonin (Sigma) and 10 uL of 10% DMSO (Sigma) or 10 uL of a 1 mMstock solution of test compound in 10% DMSO. Test compounds included:Dasatinib (BioVision), AZD5438 (Selleckchem), Staurosporine (LCLaboratories), and Nilotinib (BioVision). The plates were then incubatedfor 1 hour in a humidified, 37° C./5% CO₂ incubator. The plates wereplaced into a thermal cycler (MW Research) where the samples were heatedeither individually at different temperatures spanning a gradient of42-66° C. across the plate for 3 minutes to obtain apparent T_(agg), orthe samples are all heated for 3 minutes at one constant temperature toobtain ITDRC, followed by cooling at 22° C. for 3 minutes. The ProteinThermal Shift™ dye (Life Technologies) was added at a finalconcentration of 0.5× concentration (supplied as 1000×) in 1 uL/wellvolume. Furimazine Live Cell Substrate (Promega) was added to a finalconcentration of 1× in 20 uL/well volume, and a total volume of 100uL/well was then transferred to a tissue culture plate (Corning). Tomeasure BRET, filtered luminescence was measured on a BMG Clariostarluminometer equipped with 450 nm BP filter (donor) and 610 nm LP filter(acceptor), using 0.5s integration time. MilliBRET units (mBU) werecalculated by using the equation: (acceptor/donor)*1000. To obtainapparent T_(agg) values, data was fitted using the bell shaped curveequation. To analyze ITDFC data, data was transformed and fitted toobtain apparent EC50s using the sigmoidal dose-response (variable slope)equation. All analyses were performed with Graphpad Prism software.

FIG. 13 demonstrates that BRET can be used to detect ligand bindingthrough a change in melting temperature curves compared to DMSO controlsas exampled with CDK2-Nluc (+/−AZD5438) and LCK-Nluc (+/−Dasatinib)target fusions. As expected, the shape of the BRET curves are bellshaped due to loss of Nluc signal with protein unfolding and increasingtemperatures or dye dissociation upon protein aggregation or both.

In FIGS. 14A-B, to obtain isothermal dose response curves (ITDRC) inorder to obtain compound rank order affinity and apparent EC50s, thecurves generated when the samples were subjected to the temperaturegradient to ensure compliance with the model were inspected first,apparent Tan calculated, and an appropriate temperature chosen forfollow-up ITDRC or screening experiments. This was a temperature atwhich a majority of the protein is unfolded or precipitated in theabsence of stabilizing compound, but at which a majority of the proteinremains soluble in the presence of a saturating concentration of knownstabilizing compound. To demonstrate this, ITDRC for a panel of kinaseinhibitors with known different selectivity was established and testedfor target engagement with CDK2-Nluc and LCK-Nluc fusions as determinedby BRET after incubation in the presence of different concentrations ofthe individual compounds while time of heating and temperature were keptconstant. This demonstrates the concentration dependence of the thermalstabilization and allows for compound affinity signatures to beobtained.

Example 13 BRET-Detection Thermal Shift Assay

Experiments were conducted during development of embodiments of thepresent invention to demonstrate detection of the stabilization of atarget protein by the interaction with a target ligand via temperaturedependent alteration of BRET signal generated by the energy transferfrom a luciferase fused to the target protein to an environmentallysensitive dye that binds to all proteins unfolding in the sampleincluding the target protein.

To demonstrate analysis of compound selectivity using the BRET method ofthermal shift, a panel of compounds with known selectivity against CDK2,MAPK14, and HDAC1 was tested. It was found that the assay accuratelydetected the positive and negative ligands against these targets.

HeLa cells were transfected with CDK2-Nluc, Nuc-MAPK14, or HDAC1-NlucDNA at a 1:100 DNA ratio and incubated overnight. The cells weretrypsinized using Trypsin-EDTA (0.25%) (Gibco) and re-suspended inOpti-MEM without phenol red (Gibco)+1× Protease Inhibitor Cocktail(Promega) which had been reconstituted in DMSO (Sigma). The cells wereseeded into 96-well tall-chimney PCR plate (Fisherbrand) at a density of2×10⁴ cells/well in an 90 uL/well volume and treated with 10 uL of 10/aDMSO (Sigma) or 10 uL of a 1 mM stock solution of test compound in 10%DMSO. Test compounds included: Dasatinib (BioVision), AZD5438(Selleckchem), Staurosporine (LC Laboratories), Nilotinib (BioVision),Ponatinib (SYNKinase), AMG548 (TOCRIS), SB203580(Adipogen), SAHA(TOCRIS), Mocetinostat (Selleckchem), Panibinostat (LC Laboratories),and ACY1215 (Selleckchem). The plates were then incubated for ½ hour ina humidified, 37° C./5% CO₂ incubator. Immediately prior to heating, 10uL/well of a 0.5 mg/ml solution of digitonin (Sigma), Protein ThermalShift™ dye (Life Technologies) was added at a final concentration of0.5× concentration (supplied as 1000×) in 1 uL/well volume, andFurimazine Live Cell Substrate (Promega) was added to a finalconcentration of 1× in 20 uL/well volume. Plates were placed into athermal cycler (MW Research) where the samples were heated individuallyat different temperatures spanning a gradient of 42-66° C. across theplate for 3 minutes followed by cooling at 22° C. for 3 minutes, and atotal volume of 100 uL/well was then transferred to a tissue cultureplate (Corning). To measure BRET, filtered luminescence was measured ona BMG Clariostar luminometer equipped with 450 nm BP filter (donor) and610 nm LP filter (acceptor), using 0.5s integration time. MilliBRETunits (mBU) were calculated by using the equation:(acceptor/donor)*1000. Data was analyzed up to 62° C., and the datafitted to obtain apparent melting temperature where the protein isprecipitating (T_(agg) values) using the Boltzmann Sigmoidal equationwith Graphpad Prism software.

FIG. 15 shows detection of an increase in melting temperatures(stabilizing ligand) or no change in melting temperatures (non-binding)for cytoplasmic target fusions CDK2-Nluc and Nluc-MAPK14 as determinedby BRET after incubation with a panel of compounds, thus displayingcompound selectivity in mammalian cells subsequently exposed todigitonin and a temperature gradient. FIGS. 16A-F show detection of anincrease in melting temperatures (stabilizing ligand) or no change inmelting temperatures (non-binding) for the nuclear target fusionHDAC1-Nluc as determined by BRET after incubation with a panel ofcompounds in mammalian cells subsequently exposed to digitonin and atemperature gradient.

Example 14 BRET-Detection Thermal Shift Assay

Experiments were conducted during development of embodiments of thepresent invention to demonstrate detection of the stabilization of atarget protein by the interaction with a target ligand via temperaturedependent alteration of BRET signal generated by the energy transferfrom a luciferase fused to the target protein to an environmentallysensitive dye that binds to all proteins unfolding in the sampleincluding the target protein.

To demonstrate that many dyes are suitable for the luciferase-basedthermal shift assay analyzed by BRET described herein, several differentdyes were tested. Dye examples included: Protein Thermal Shift™ Dye,SYPRO Orange protein gel stain, and SYPRO Red protein gel stain.

HeLa cells were transfected with CDK2-Nluc at a 1:100 DNA ratio andincubated overnight. The cells were trypsinized using Trypsin-EDTA(0.25%) (Gibco) and re-suspended in Opti-MEM without phenol red(Gibco)+1× Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well inan 90 uL/well volume and treated with 10 uL of 10% DMSO (Sigma) or 10 uLof a 1 mM stock solution of Staurosporine (LC Laboratories) in 10% DMSO.The plates were then incubated for ½ hour in a humidified, 37° C./5% CO₂incubator. Immediately prior to heating, 10 uL/well of a 0.5 mg/mlsolution of digitonin (Sigma), Furimazine Live Cell Substrate (Promega)to a final concentration of 1× in 20 uL/well volume, and 1 ul of eachdye dilution was added. Dyes included the SYPRO Orange, SYPRO Red, andPROTEIN THERMAL SHIFT Dye (all from Life Tech). Plates were placed intoa thermal cycler (MW Research) where the samples were heatedindividually at different temperatures spanning a gradient of 42-66° C.across the plate for 3 minutes followed by cooling at 22° C. for 3minutes, and a total volume of 100 uL/well was then transferred to atissue culture plate (Corning). To measure BRET, filtered luminescencewas measured on a BMG Clariostar luminometer equipped with 450 nm BPfilter (donor) and 610 nm LP filter (acceptor), using 0.5s integrationtime. MilliBRET units (mBU) were calculated by using the equation:(acceptor/donor)*1000. Data was then fitted to obtain apparent meltingtemperature where the protein is precipitating (T_(agg) values) usingthe Boltzmann Sigmoidal equation with Graphpad Prism software.

FIGS. 17A-B show detection of an increase in melting temperature forCDK2-Nluc as determined by BRET after incubation with stabilizing ligand(staurosporine) in mammalian cells subsequently exposed to digitonin anda temperature gradient using three different environmentally sensitiveacceptor dyes reporting on protein folding status as BRET acceptor dyes.B_(max) is acceptor dye dose dependent, but that there is no change inthe apparent melting temperature (T_(agg)).

Example 15 BRET-Detection Thermal Shift Assay

Experiments were conducted during development of embodiments of thepresent invention to demonstrate detection of the stabilization of atarget protein by the interaction with a target ligand via temperaturedependent alteration of BRET signal generated by the energy transferfrom a luciferase fused to the target protein to an environmentallysensitive dye that binds to all proteins unfolding in the sampleincluding the target protein.

To demonstrate that the acceptor dye can be added either before or afterthe heating step in the luciferase-based thermal shift assay using BRETanalysis, two different environmentally sensitive dyes were used todetermine folded protein status and serve as BRET acceptor dyes. Thedyes included in the Protein Thermal Shift™ Dye (Life Technologies) andthe PROTEOSTAT dye (Enzo), which have differing properties in regards toprotein status binding. The PROTEOSTAT dye binds to protein aggregatesrather than unfolding proteins, whereas the Protein Thermal Shift™ dyebinds to proteins as they unfold, but dissociates as the proteinsaggregate. (FIG. 18 ).

HeLa cells were transfected with CDK2-Nluc DNA at a 1:10 DNA ratio andincubated overnight. The cells were trypsinized using Trypsin-EDTA(0.25%) (Gibco) and re-suspended in Opti-MEM without phenol red(Gibco)+1× Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well ina 90 uL/well volume and treated with 10 uL/well of a 0.5 mg/ml solutionof digitonin (Sigma) in 10% DMSO (Sigma) and 1 uL of 100% DMSO (Sigma)or 1 uL of a 10 mM stock solution of Staurosporine (LC Laboratories) in100% DMSO. The plates were then incubated for 2 hour in a humidified,37° C./5% CO₂ incubator.

The plates were placed into a thermal cycler (MW Research) where thesamples were heated either individually at different temperaturesspanning a gradient of 42-66° C. across the plate for 3 minutes followedby cooling at 22° C. for 3 minutes. The Protein Thermal Shift™ Dye (LifeTechnologies) and the PROTEOSTAT dye (Enzo) were added at a finalconcentration of 1× (supplied as 1000×) in 1 uL/well volume eitherbefore or after the heating step. 90 uL/well was transferred to a tissueculture plate (Corning), and Furimazine Live Cell Substrate (Promega)was added to a final concentration of 1× in 20 uL/well volume. Tomeasure BRET, filtered luminescence was measured on a BMG Clariostarluminometer equipped with 450 nm BP filter (donor) and 610 nm LP filter(acceptor), using 0.5s integration time. MilliBRET units (mBU) werecalculated by using the equation: (acceptor/donor)*1000. Data was fittedusing the bell shaped curve equation with Graphpad Prism software. (FIG.18 )

Example 16 BRET-Detection Thermal Shift Assay

Experiments were conducted during development of embodiments of thepresent invention to demonstrate detection of the stabilization of atarget protein by the interaction with a target ligand via temperaturedependent alteration of BRET signal generated by the energy transferfrom a luciferase fused to the target protein to an environmentallysensitive dye that binds to all proteins unfolding in the sampleincluding the target protein.

HeLa cells were transfected with CDK2-Nluc or HDAC1-Nluc at a 1:100 DNAratio and incubated overnight. The cells were trypsinized usingTrypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM without phenolred (Gibco)+1× Protease Inhibitor Cocktail (Promega) which had beenreconstituted in DMSO (Sigma). The cells were seeded into 96-welltall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴ cells/well inan 80 uL/well volume and treated with 10 uL of 10% DMSO or 10 uL of a 1mM stock solution of AZD5438 (for CDK2-Nuc) or Mocetinostat (forHDAC1-Nluc) in 10% DMSO. The plates were then incubated for ½ hour in ahumidified, 37° C./5% CO₂ incubator. Immediately prior to heating, 10uL/well of a 0.5 mg/ml solution of digitonin (Sigma), Furimazine LiveCell Substrate (Promega) to a final concentration of 1× in 20 uL/wellvolume, and 1 ul of the Protein Thermal Shift™ Dye (Life Technologies)for a final concentration of 0.5× was added. Plates were placed into athermal cycler (MW Research) where the samples were heated individuallyat different temperatures spanning a gradient of 42-66° C. across theplate for 3 minutes. Some plates were also then further cooled at 22° C.for 3 minutes. A total volume of 100 uL/well was then transferred to atissue culture plate (Corning). To measure BRET, filtered luminescencewas measured on a BMG Clariostar luminometer equipped with 450 nm BPfilter (donor) and 610 nm LP filter (acceptor), using 0.5s integrationtime. MilliBRET units (mBU) were calculated by using the equation:(acceptor/donor)*1000. Data was then fitted to obtain apparent meltingtemperature where the protein is precipitating (Ta, values) using theBoltzmann Sigmoidal equation with Graphpad Prism software.

FIGS. 19 and 20A-B demonstrate data obtained when the assay was run withand without the cooling step that follows the 3 minutes heating gradientstep using two different targets, CDK2-Nluc (FIG. 19 ) and HDAC1-Nluc(FIGS. 20A-B). A clear shift in apparent Tan was found whether a coolingstep was included in the protocol or not as analyzed by both, directluciferase readout and BRET.

Example 17 Other Proposed Proximity Based Reporter Methods for Detectionof Ligand Binding in a Thermal Shift Assay

Based on the successful use of a direct luciferase fusion or BRET todetect ligand-mediated thermal stabilization, other proximity-basedreporter chemistries are understood to be useful. FIG. 21 givesschematic examples of other proximity based reporter methods that may besuitable for analysis of target engagement using thermal shift. Forexample, an epitope tag is attached to the protein of interest andfollowing a thermal denaturation step, addition of a detection antibodylabeled with a donor fluorophore (e.g., terbium, europium, etc.) is usedin a FRET assay with an denaturation/aggregation-sensitive dye as a FRETacceptor. Ligand-mediated thermal stabilization results in a loss ofFRET/TR-FRET signal. Another potential iteration is using a combinationof labeled antibodies (e.g. donor-labeled anti-FLAG and acceptor labeledanti-V5) and a tandem epitope (e.g. FLAG-V5) tethered to the targetprotein are used as a detection system. When the target is stabilized bybinding of a ligand, the epitope is presented and both antibodies bind,generating a proximity-based signal (e.g. FRET, TR-FRET). Upon thermaldenaturation, the epitopes are unavailable to the antibody pair,resulting in a loss of proximity-based signal. Various detectionchemistries can be applied for these prophetic examples (e.g. TR-FRET,proximity ligation, singlet oxygen transfer/alphascreen, etc.).

Example 18 Chemical Synthesis Description

A series of styryl dye derivatives were synthesized and screened in BRETdetection thermal shift assay. The dye derivatives CS000, CS0004,CS0007, CS0008, CS0013, CS0010, CS0017, CS0018, CS0020, CS0024, CS0028,CS0038, CS0067, CS0068, CS0075, CS0081, CS0085, CS0086, CS0087, CS0096,and CS0155 and are generally synthesized analogously to CS0101 via acondensation reaction between various aryl aldehydes and pyridiniumbetaines or N-alkyl pyridinium, which has been described in literature(1^(st) reaction in Scheme 1) (Hassner, A.; Birnbaum, D.; Loew, L. M. J.Org. Chem. 1984, 49, 2546-2551.; herein incorporated by reference in itsentirety). As depicted in Scheme 1, CS0101 was hydrolyzed under acidiccondition to afford CS0036. The subsequent amide coupling and the acidicdeprotection afforded CS0100 and CS0045 respectively. CS0048 wasobtained via an amide coupling between CS0045 and3-amino-1-propanesulfonic acid. CS0043 and CS0147 was synthesizedanalogously to CS0045. CS0073 was obtained via a N-alkylation reactionbetween CS0043 and neat 1,3 propane sultone (Scheme 2). CS0117 wassynthesized via a Heck reaction between the para-bromo aniline 5 andpara-vinyl pyridine 6 (Scheme 3).¹ N-alkylation of CS0117 affordedCS0121 (Scheme 3).¹ CS0112, CS0158, CS0071 and CS0072 were synthesizedanalogously to CS0121.

(E)-4-(4-(Dihexylamino)styryl)-1-(4-ethoxy-4-oxobutyl)pyridin-1-iumbromide (CS0101): To a solution of 4-(dihexylamino)benzaldehyde (1, 144mg, 0.5 mmol, 1.0 equiv) in EtOH (4 mL) at RT, piperidine (4.3 mg, 0.05mmol. 0.1 equiv) and 1-(4-ethoxy-4-oxobutyl)-4-methylpyridin-1-iumbromide (2, 144 mg, 0.5 mmol, 1.0 equiv) was added in one portion. Themixture was stirred at 90° C. for 15 h and cooled down at RT for 30 min.The desired product precipitated from the solution and was purified bysilica gel chromatography (CH₂Cl₂/MeOH, 1/9). LRMS: observed for[M−Br]⁺, 479.7.

(E)-1-(3-Carboxypropyl)-4-(4-(dihexylamino)styryl)pyridin-1-ium chloride(CS0036): To a solution of CS0101 (324 mg, 0.58 mmol, 1.0 equiv) in1,4-dioxane (4 mL) at RT, 6 N HCl (aq, 4 mL, 24 mmol, 41 equiv) wasadded. The mixture was stirred at 100° C. for 15 h and concentrated invacuo to afford the crude. The desired product was purified by silicagel chromatography (CH₂Cl₂/MeOH, 1/9). LRMS: observed for [M−Cl]⁺,451.7.

(E)-4-(4-(Dihexylamino)styryl)-1-(2,2-dimethyl-4,17-dioxo-3,7,10,13-tetraoxa-16-azaicosan-20-yl)pyridin-1-iumchloride (CS0100): To a solution of CS0036 (70 mg, 0.13 mmol, 1.0 equiv)in DMF (1.5 mL) at RT, TSTU (40 mg, 0.13 mmol, 1.0 equiv) and NEt₃ (60μL, 0.43 mmol, 2.5 equiv) was added. The solution was stirred at RT for1 h. To the reaction mixture, tert-butyl3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (3, 43.8 mg, 0.16 mmol,1.2 equiv) in DMF (1.5 mL) was added. The reaction was stirred at RT for15 h. LC-MS analysis indicated full conversion of the starting material.The solvent was removed in vacuo to afford the crude and the desiredproduct was purified by silica gel chromatography (CH₂Cl₂/MeOH, 1/9).LRMS: observed for [M−Cl]⁺, 711.1.

(E)-1-(1-Carboxy-13-oxo-3,6,9-trioxa-12-azahexadecan-16-yl)-4-(4-(dihexylamino)styryl)pyridin-1-iumchloride (CS0045): The starting material CS0100 (40 mg, 0.5 mmol, 1.0equiv) was dissolved in 4N HCl in dioxane (2 mL, 8 mmol, 16 equiv). Themixture was stirred at 100° C. for 15 h and concentrated in vacuo toafford the crude. The desired product was purified by silica gelchromatography (CH₂Cl₂/MeOH, 1/9). LRMS: observed for [M−Cl]⁺, 654.9.

(E)-21-(4-(4-(Dihexylamino)styryl)pyridin-1-ium-1-yl)-5,18-dioxo-8,11,14-trioxa-4,17-diazahenicos-ane-1-sulfonate(CS0948): To a solution of CS0045 (60 mg, 0.09 mmol, 1.0 equiv) in DMF(1.5 mL) at RT, TSTU (33 mg, 0.11 mmol, 1.2 equiv) and NEt₃ (51 μL, 0.37mmol, 4 equiv) was added. The solution was stirred at RT for 1 h. To thereaction mixture, 3-amino-1-propanesulfonic acid (4, 19.1 mg, 0.14 mmol,1.5 equiv) in H₂O (1 mL) was added. The reaction was stirred at RT for15 h. LC-MS analysis indicated full conversion of the starting material.The solvent was removed in vacuo to afford the crude and the desiredproduct was purified by silica gel chromatography (CH₂Cl₂/MeOH, 1/9).LRMS: observed for [M+H]⁺, 776.1.

(E)-3-(4-(4-(4-(Dihexylamino)styryl)pyridin-1-ium-1-yl)-N-(3-sulfopropyl)butanamido)propane-1-sulfonate(CS0073): CS0043 was mixed with 1,3 propane sultone (1 mL) and heated at120° C. for 3 h. The mixture was cooled down to RT and resuspended inhot MeOH (20 mL), filtered over Celite. Concentration in vacuo affordedthe crude, which was purified by silica gel chromatography (CH₂Cl₂/MeOH,1/9). LRMS: observed for [M+H]⁺, 695.1.

(E)-N,N-Dihexyl-4-(2-(pyridin-4-y)vinyl)aniline (CS0117): To a flaskcharged with Pd(OAc)₂ (11.2 mg, 50 μL, 0.05 equiv) and tri-(o-tolyl)phosphine (45.6 mg, 0.14 mmol, 0.15 equiv), NEt₃ (5 mL) was added. Themixture was stirred under N2 for 20 min. To the mixture, para-bromoaniline (5, 340 mg, 1 mmol, 1.0 equiv) and para-vinyl pyridine (6, 115.7mg, 1.1 mmol, 1.1 equiv) was added. The mixture was heated up to 120° C.and stirred for 48 h under N2. The reaction was cooled down andconcentrated to afford the crude. The desired product was purified bysilica gel chromatography (Heptane/EtOAc, 6/4). LRMS: observed for[M+H]⁻, 365.6.

(E)-4(4-(dihexylamino)styryl)-1-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)pyridin-1-iumiodide(C30121): To a solution of CS0117 (70 mg, 0.19 mmol, 1.0 equiv) in CH₃CN(3 mL), 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluoro-8-iodooctane (7, 108 mg,0.23 mmol, 1.2 equiv) was added. The reaction was heated to reflux for15 h. LC-MS indicated full conversion to the desired product. Themixture was concentrated in vacuo and purified by silica gelchromatography (CH₂Cl₂/MeOH, 1/9). LRMS: observed for [M−I]⁺, 711.8.

Example 19 BRET-Detection Thermal Shift Assay

Experiments were conducted during development of embodiments of thepresent invention to demonstrate detection of the stabilization of atarget protein by the interaction with a target ligand via temperaturedependent alteration of BRET signal generated by the energy transferfrom a luciferase fused to the target protein to an environmentallysensitive dye that binds to all proteins unfolding in the sampleincluding the target protein.

To demonstrate analysis of compound selectivity using the BRET method ofthermal shift, a panel of compounds with known selectivity against CDK2and MAPKI4 was tested. It was found that the assay accurately detectedthe positive and negative ligands against these targets.

HeLa cells were transfected with CDK2-Nluc or Nluc-MAPKI4 DNA at a 1:10DNA ratio and incubated overnight. The cells were trypsinized usingTrypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM without phenolred (Gibco)+1× Protease Inhibitor Cocktail (Promega), which had beenreconstituted in DMSO (Sigma). The cells were seeded into wells of a96-well, tall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴cells/well in an 90 uL/well volume and treated with 10 uL of 10% DMSO(Sigma) or 10 uL of a 1 mM stock solution of the test compoundStaurosporine (LC Laboratories) in 10% DMSO. The plates were thenincubated for ½ hour in a humidified, 37° C./5% CO₂ incubator.Immediately prior to heating, 10 uL/well of a 0.5 mg/ml solution ofdigitonin (Sigma) and dye derivative was added at a final concentrationof 10 uM or in a dose response as indicated in B and C of each figure in1 uL/well volume. Plates were placed into a thermal cycler (MW Research)where the samples were heated individually at different temperaturesspanning a gradient of 42-66° C. across the plate for 3 minutes followedby cooling at 22° C. for 3 minutes, and a total volume of 100 uL/wellwas then transferred to a tissue culture plate (Corning). FurimazineLive Cell Substrate (Promega) was added to a final concentration of 1×in 20 uL/well volume. To measure BRET, filtered luminescence wasmeasured on a BMG Clariostar luminometer equipped with 450 nm BP filter(donor) and 610 nm LP filter (acceptor), using 0.5s integration 5 time.MilliBRET units (mBU) were calculated by using the equation:(acceptor/donor)*1000.

Data was analyzed up to 62° C., and the data fitted to obtain apparentmelting temperature where the protein is precipitating (Tagg values)using the Boltzmann Sigmoidal equation with Graphpad Prism software.

FIGS. 23-34 demonstrate: (A) that BRET using dyes synthesized in Example18 detects ligand binding through a change in relative BRET compared toDMSO controls; (B) the fold change in BRET signal at 56° C. over thebackground BRET signal at 42° C. for cells transfected with CDK2-Nluctarget that were treated with DMSO and varying dye concentrations; and(C) the fold change in BRET signal at 52° C. over the background BRETsignal at 42° C. for cells transfected with Nluc-MAPK14 target that weretreated with DMSO and varying dye concentrations

Example 20

The properties of environmentally sensitive dyes for thermal shiftassays include having a gain in fluorescence upon binding to proteinsthat are unfolding due to thermal insult. FIGS. 35-37 demonstrate thefluorescent spectra for some of the synthesized dyes (described inExample 18) when added to cells lysates that had been transfected withNluc-target protein, and then either heated at 42° C. (baseline), 56° C.(for CDK2-Nluc target), or 52° C. (for Nluc-MAPK14 target). The highertemperature results in a gain in fluorescence thus showing the change indye binding and fluorescent properties indicating that it is a usefuldye for this application.

HeLa cells were transfected with CDK2-Nluc or Nluc-MAPKI4 DNA at a 1:10DNA ratio and incubated overnight. The cells were trypsinized usingTrypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM without phenolred (Gibco)+1× Protease Inhibitor Cocktail (Promega), which had beenreconstituted in DMSO (Sigma). The cells were seeded into wells of a96-well, tall-chimney PCR plate (Fisherbrand) at a density of 2×10⁴cells/well in an 90 uL/well volume and treated with 10 uL of 10× dyesolution and heated at 42° C., 52° C., or 56° C. for 3 minutes followedby cooling at 25° C. for 2 minutes. Samples were transferred to a tissueculture plate and analyzed in fluorescent spectral mode on a Clariostar.

Example 21 Target Genome Editing

Targeted genome editing technologies to introduce reporter tags to atarget protein of interest are used to evaluate ligand binding inducedthermal stabilization at endogenous levels of protein expression in therelevant cellular context. Targeted genome editing technologies include,but are not limited to, clustered regularly-interspaced shortpalindromic repeats (CRISPR/Cas system), zinc finger nucleases (ZFN),transcription-activator-like effector-based nucleases (TALENs), andengineered meganucleases re-engineered homing endonucleases. Targetedprotein fusions are made, in some embodiments, with a high affinityNLpoly, NLpoly, or NANOLUC luciferase to a protein of interest. Thesetags are fused to the N- or C-terminal of the protein of interest orinternally within the target protein of interest. Assays are performedin both selected clonal cell lines as well as in unselected pools ofcells. These assays are performed under live cell or lytic conditions.Assays in which the peptide reporters are used (high affinity NLpoly andNLpoly) require that the complementary peptide (i.e., NLpep) be presentfor reconstitution of active reporter luciferase and signal. In someembodiments, this is done lytically using a reagent that contains thecomplementary NLpep or through using mammalian cells that expresses theNLpep complementary to the NLpep used for the genomic editing of thetarget of interest.

SEQUENCES (wild-type OgLuc)- SEQ ID NO: 1mftladfvgdwqqtagynqdqvleqgglsslfgalgvsvtpiqkvvlsgenglkadihviipyeglsgfqmgliemifkvvypvddhhfkiilhygtividgvtpnmidyfgrpypgiavfdgkqitvtgtlwngnkiyderlinpdgsl lfrvtingvtgwrlcenila(NANOLUC)- SEQ ID NO: 2mvftledfvgdwrqtagynldqvleqggvsslfqnlgvsvtpiqrivlsgenglkidihviipyeglsgdqmgqiekifkvvypvddhhfkvilhygtividgvtpnmidyfgrpyegiavfdgkkitvtgtlwngnkiiderlinpdgs llfrvtingvtgwrlcerilaSEQ ID NO: 3 gvtgwrlcerila SEQ ID NO: 4mvftladfvgdwqqtagynqdqvleqgglsslfgalgvsvtpiqkvvlsgenglkadihviipyeglsgfqmgliemifkvvypvddhhfkiilhygtividgvtpnmidyfgrpypgiavfdgkqitvtgtlwngnkiyderlinpdgs llfrvtin (NLpep86)-SEQ ID NO: 5 vsgwrlfkkis (NLpoly11S)- SEQ ID NO: 6mvftledfvgdweqtaaynldqvleqggvssllqnlaysvtpiqrivrsgenalkidihviipyeglsadqmaqieevfkvvypvddhhfkvilpygtividgvtpnmlnyfgrpyegiavfdgkkitvtgtlwngnkiiderlitpdgs mlfrvtins(NLpoly156)- SEQ ID NO: 7mvftledfvgdwrqtagynldqvleqggvsslfqnlgvsvtpiqrivlsgenalkidihviipyeglsgdqmgqiekifkvvypvddhhfkvilhygtividgvtpnmidyfgrpyegiavfdgkkitvtgtlwngnkiiderlinpdgs llfrvtin

All publications and patents provided herein are incorporated byreference in their entireties. Various modifications and variations ofthe described compositions and methods of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thepresent invention.

The invention claimed is:
 1. A system comprising: (a) a fusion of a target protein and a peptide tag comprising 70% or greater sequence identity with VSGWRLFKKIS (SEQ ID NO: 5), (b) a complementary polypeptide comprising 70% or greater sequence identity with SEQ ID NO: 6, wherein the peptide tag and the complementary polypeptide are capable of forming a bioluminescent complex; and (c) a fluorescent dye that: (1) binds nonspecifically to aggregated proteins and/or hydrophobic peptide segments, and (2) has an excitation spectrum that overlaps with the emission spectrum of the bioluminescent complex.
 2. The system of claim 1, wherein the fluorescent dye is capable of being quenched by water.
 3. The system of claim 1, wherein the fluorescent dye is fluorogenic.
 4. The system of claim 1, wherein the system is a cell, a cell lysate, or a reaction mixture.
 5. The system of claim 1, further comprising a ligand of the target protein.
 6. A method to detect an interaction between a ligand and a target protein, comprising the steps of: (a) incubating the system of claim 1 with a substrate for the bioluminescent complex: (i) in the presence of the ligand to produce a test sample, and (ii) in the absence of the ligand, to produce a control sample; (b) treating said test and control samples under conditions that cause the target protein to partially unfold; (c) measuring signal from the fluorescent dye in said test and control samples; and (d) comparing the measurement made in step (c) between the test and control samples, wherein alteration of the signal from said fluorescent dye in the test sample compared to the control sample indicates the presence of the interaction between the ligand and the target protein.
 7. The method of claim 6, wherein the system of claim 1 is within a cell, a cell lysate, or a reaction mixture.
 8. The method of claim 7, wherein the ligand is added exogenously to the cell, the cell lysate, or the reaction mixture.
 9. The method of claim 6, wherein the fusion and/or complementary polypeptide are expressed within the cell, the cell lysate, or the reaction mixture.
 10. The method of claim 6, wherein the conditions that cause the target protein to partially unfold comprise elevated temperature and/or a denaturant.
 11. The method of claim 10, elevated temperature comprises one or more temperatures above physiologic temperature.
 12. The method of claim 10, elevated temperature comprises one or more temperatures near the approximate melting temperature of the target protein.
 13. The method of claim 6, wherein a plurality of test samples are produced using a plurality of test ligands. 